NOVEL ANTI-cMET ANTIBODY

ABSTRACT

The invention relates to a novel antibody capable of binding specifically to the human c-Met receptor and/or capable of specifically inhibiting the tyrosine kinase activity of said receptor both in a ligand-dependent and in a ligand-independent manner, with an improved antagonistic activity, said antibody comprising a modified hinge region. 
     The invention also relates to a composition comprising such an antibody antagonist to c-Met and its use as a medicament for treating cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/EP2009/066201, filedon Dec. 2, 2009, that designates the United States; and claims thebenefit under 35 U.S.C §119(e) of U.S. provisional patent applicationNo. 61/184,502, filed Jun. 5, 2009, and the benefit under 35 U.S.C.§365(b) of PCT/IB2008/055663, filed Dec. 2, 2008, that designates atleast one country other than the United States; all of which areincorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“SequenceListing.txt”, created on Jun. 1, 2010, and having a size of 138kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

The present invention relates to a novel divalent antibody capable ofbinding specifically to the human c-Met receptor and/or capable ofspecifically inhibiting the tyrosine kinase activity of said receptor,as well as the amino acid and nucleic acid sequences coding for saidantibody. More particularly, the antibody according to the invention iscapable of inhibiting the c-Met dimerization. The invention likewisecomprises the use of said antibody as a medicament for the prophylacticand/or therapeutic treatment of cancers or any pathology connected withthe overexpression of said receptor as well as in processes or kits fordiagnosis of illnesses connected with the over-expression of c-Met. Theinvention finally comprises products and/or compositions comprising suchas antibody in combination with other antibodies and/or chemicalcompounds directed against other growth factors involved in tumorprogression or metastasis and/or compounds and/or anti-cancer agents oragents conjugated with toxins and their use for the prevention and/orthe treatment of certain cancers.

Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab,cetuximab, bevacizumab, imatinib and gefitinib inhibitors haveillustrated the interest of targeting this protein class for treatmentof selected cancers.

c-Met, is the prototypic member of a sub-family of RTKs which alsoincludes RON and SEA. The c-Met RTK family is structurally differentfrom other RTK families and is the only known high-affinity receptor forhepatocyte growth factor (HGF), also called scatter factor (SF) [D. P.Bottaro et al., Science 1991, 251:802-804; L. Naldini et al., Eur. Mol.Biol. Org. J. 1991, 10:2867-2878]. c-Met and HGF are widely expressed ina variety of tissue and their expression is normally restricted to cellsof epithelial and mesenchymal origin respectively [M. F. Di Renzo etal., Oncogene 1991, 6:1997-2003; E. Sonnenberg et al., J. Cell. Biol.1993, 123:223-235]. They are both required for normal mammaliandevelopment and have been shown to be particularly important in cellmigration, morphogenic differentiation, and organization of thethree-dimensional tubular structures as well as growth and angiogenesis[F. Baldt et al., Nature 1995, 376:768-771; C. Schmidt et al., Nature,1995: 373:699-702; Tsarfaty et al., Science 1994, 263:98-101]. While thecontrolled regulation of c-Met and HGF have been shown to be importantin mammalian development, tissue maintenance and repair [Nagayama T.,Nagayama M., Kohara S., Kamiguchi H., Shibuya M., Katoh Y., Itoh J.,Shinohara Y., Brain Res. 2004, 5;999(2):155-66; Tahara Y., Ido A.,Yamamoto S., Miyata Y., Uto H., Hori T., Hayashi K., Tsubouchi H., JPharmacol Exp Ther. 2003, 307(1):146-51], their dysregulation isimplicated in the progression of cancers.

Aberrant signalling driven by inappropriate activation of c-Met is oneof the most frequent alteration observed in human cancers and plays acrucial role in tumorigenesis and metastasis [Birchmeier et al., Nat.Rev. Mol. Cell Biol. 2003, 4:915-925; L. Trusolino and Comoglio P. M.,Nat Rev. Cancer. 2002, 2(4):289-300].

Inappropriate c-Met activation can arise by ligand-dependent andindependent mechanisms, which include overexpression of c-Met, and/orparacrine or autocrine activation, or through gain in function mutation[J. G. Christensen, Burrows J. and Salgia R., Cancer Latters. 2005,226:1-26]. However an oligomerization of c-Met receptor, in presence orin absence of the ligand, is required to regulate the binding affinityand binding kinetics of the kinase toward ATP and tyrosine-containingpeptide substrates [Hays J L, Watowich S J, Biochemistry, 2004 Aug. 17,43:10570-8]. Activated c-Met recruits signalling effectors to itsmultidocking site located in the cytoplasm domain, resulting in theactivation of several key signalling pathways, including Ras-MAPK, PI3K,Src and Stat3 [Gao C F, Vande Woude G F, Cell Res. 2005, 15(1):49-51;Furge K A, Zhang Y W, Vande Woude G F, Oncogene. 2000, 19(49):5582-9].These pathways are essential for tumour cell proliferation, invasion andangiogenesis and for evading apoptosis [Furge K A, Zhang Y W, VandeWoude G F, Oncogene, 2000, 19(49):5582-9; Gu H., Neel B G, Trends CellBiol. 2003 Mar. 13(3):122-30; Fan S., Ma Y X, Wang J A, Yuan R Q, MengQ., Cao Y., Laterra J J. Goldberg I D, Rosen E M, Oncogene. 2000 Apr.27, 19(18):2212-23]. In addition, a unique facet of the c-Met signallingrelative to other RTK its reported interaction with focal adhesioncomplexes and non kinase binding partners such as α6β4 integrins[Trusolino L., Bertotti A., Comoglio P M, Cell. 2001, 107:643-54],CD44v6 [Van der Voort R., Taher T E, Wielenga V J, Spaargaren M., PrevoR., Smit L., David G., Hartmann G., Gherardi E., Pals S T. J. Biol.Chem. 1999, 274(10):6499-506]. Plexin B1 or semaphorins [Giordano S.,Corso S., Conrotto P., Artigiani S., Gilestro G., Barberis D., TamagnoneL., Comoglio P M, Nat Cell Biol. 2002, 4(9):720-4; Conrotto P.,Valdembri D., Corso S., Serini G., Tamagnone L., Comoglio P M, BussolinoF., Giordano S., Blood. 2005, 105(11):4321-9; Conrotto P., Corso S.,Gamberini S., Comoglio P M, Giordano S., Oncogene. 2004, 23:5131-7]which may further add to the complexity of regulation of cell functionby this receptor. Finally recent data demonstrate that c-Met could beinvolved in tumor resistance to gefitinib or erlotinib suggesting thatcombination of compound targeting both EGFR and c-Met might be ofsignificant interest [Engelman J A et al., Science, 2007, 316:1039-43].

In the past few years, many different strategies have been developed toattenuate c-Met signalling in cancer cell lines. These strategiesinclude i) neutralizing antibodies against c-Met or HGF/SF [Cao B., SuY., Oskarsson M., Zhao P., Kort E J, Fisher R J, Wang L M, Vande Woude GF, Proc Natl Acad Sci U S A. 2001, 98(13):7443-8; Martens T., Schmidt NO, Eckerich C., Fillbrandt R., Merchant M., Schwall R., Westphal M.,Lamszus K., Clin Cancer Res. 2006, 12(20):6144-52] or the use of HGF/SFantagonist NK4 to prevent ligand binding to c-Met [Kuba K., MatsumotoK., Dale K., Shimura H., Tanaka M., Nakamura T., Cancer Res., 2000,60:6737-43], ii) small ATP binding site inhibitors to c-Met that blockkinase activity [Christensen J G, Schreck R., Burrows J., Kuruganti P.,Chan E, Le P., Chen J., Wang X., Ruslim L., Blake R., Lipson K E,Ramphal J., Do S., Cui J J, Cherrington J M, Mendel D B, Cancer Res.2003, 63:345-55]; iii) engineered SH2 domain polypeptide that interfereswith access to the multidocking site and RNAi or riboxyme that reducereceptor or ligand expression. Most of these approaches display aselective inhibition of c-Met resulting in tumor inhibition and showingthat c-Met could be of interest for therapeutic intervention in cancer.

Within the molecules generated for c-Met targeting, some are antibodies.The most extensively described is the anti-C-Met 5D5 antibody generatedby Genentech [WO 96/38537] which behaves as a potent agonist when addedalone in various models and as an antagonist when used as a Fabfragment. A monovalent engineered form of this antibody described as onearmed 5D5 (OA5D5) and produced as a recombinant protein in E. Coli isalso the subject of a patent application [WO 2006/015371] by Genentech.However, this molecule that could not be considered as an antibodybecause of its particular scaffold, displays also mutations that couldbe immunogenic in humans. In terms of activity, this unglycosylatedmolecule is devoided of effector functions and finally, no clear datademonstrate that OA5D5 inhibits dimerization of c-Met. Moreover, whentested in the G55 in vivo model, a glioblastoma cell line that expressesc-Met but not HGF mRNA and protein and that grows independently of theligand, the one armed anti-c-Met had no significant effect on G55 tumorgrowth suggesting that OA5D5 acts primarily by blocking HGF binding andis not able to target tumors activated independently of HGF [Martens T.et al, Clin. Cancer Res., 2006, 12(20):6144-6152].

Another antibody targeting c-Met is described by Pfizer as an antibodyacting “predominantly as c-Met antagonist, and in some instance as ac-Met agonist” [WO 2005/016382]. No data showing any effect of Pfizerantibodies on c-Met dimerization is described in this application.

One of the innovative aspects of the present invention is to generate achimeric and/or humanized monoclonal antibody without intrinsic agonistactivity and inhibiting c-Met dimerization. More particularly, aninnovative aspect of the present invention is to generate a chimericand/or humanized monoclonal antibody with antagonist activity andinhibiting c-Met dimerization.

In addition of targeting ligand-dependent tumors, this approach willalso impair ligand-independent activations of c-Met due to itsoverexpression or mutations of the intra cellular domain which remaineddependent to oligomerization for signalling. Another aspect of theactivity of this antibody could be a steric hindrance for c-Metinteraction with its partners that will result in impairment of c-Metfunctions. This antibody is humanized and engineered preferentially, butnot limited, human IgG1 to get effector functions such as ADCC and CDCin addition to functions linked to the specific blockade of the c-Metreceptor.

Surprisingly, for the first time, inventors have managed to generate achimeric and/or humanized monoclonal antagonist antibody capable ofbinding to c-Met but also capable of inhibiting the c-Met dimerization,said monoclonal antibody being divalent contrary to existing antagonistantibodies directed against c-Met. If it is true that, in the prior art,it is sometimes suggested that an antibody capable of inhibiting thedimerization of c-Met with its partners could be an interesting one, ithas never been disclosed, or clearly suggested, an antibody capable ofdoing so. Moreover, regarding antibody specificity, it was not evidentat all to succeed in the generation of such an active divalent antibody.

As it was explained before, the inhibition of the c-Met dimerization isa capital aspect of the invention as such antibodies will present a realinterest for a larger population of patients. Not only ligand-dependentactivated c-Met cancer, as it was the case until the present invention,but also ligand-independent activated c-Met cancer could be traded withantibodies generated by the process of the present invention.

Antibodies were evaluated by BRET analysis on cells expressing bothc-Met-RLuc/c-Met-YFP and selected on their ability to inhibit at least40% preferably 45%, 50%, 55% and most preferably 60% of the BRET signal.

The BRET technology is known as being representative of the proteindimerization [Angers et al., PNAS, 2000, 97:3684-89].

The BRET technology is well known by the man skill in the art and willbe detailed in the following examples. More particularly, BRET(Bioluminescence Resonance Energy Transfer) is a non-radiative energytransfer occurring between a bioluminescent donor (Renilla Luciferase(Rluc)) and a fluorescent acceptor, a mutant of GFP (Green FluorescentProtein) or YFP (Yellow fluorescent protein). In the present case EYFP(Enhanced Yellow Fluorescent Protein) was used. The efficacy of transferdepends on the orientation and the distance between the donor and theacceptor. Then, the energy transfer can occur only if the two moleculesare in close proximity (1-10 nm). This property is used to generateprotein-protein interaction assays. Indeed, in order to study theinteraction between two partners, the first one is genetically fused tothe Renilla Luciferase and the second one to the yellow mutant of theGFP. Fusion proteins are generally, but not obligatory, expressed inmammalian cells. In presence of its membrane permeable substrate(coelenterazine), Rluc emits blue light. If the GFP mutant is closerthan 10 nm from the Rluc, an energy transfer can occur and an additionalyellow signal can be detected. The BRET signal is measured as the ratiobetween the light emitted by the acceptor and the light emitted by thedonor. So the BRET signal will increase as the two fusion proteins arebrought into proximity or if a conformational change brings Rluc and GFPmutant closer.

If the BRET analysts consists in a preferred embodiment, any methodknown by the man skilled in the art can be used to measure c-Metdimerization. Without limitation, the following technologies can bementioned; FRET (Fluorescence Resonance Energy Transfer), HTRF(Homogenous Time resolved Fluorescence), FLIM (Fluorescence LifetimeImaging Microscopy) or SW-FCCS single wavelength fluorescencecross-correlation spectroscopy).

Other classical, technologies could also be used, such asCo-immunoprecipitation, Alpha screen, Chemical cross-linking,Double-Hybrid, Affinity Chromatography, ELISA or Far western blot.

The terms “antibody”, “antibodies” or “immunoglobulin” are usedinterchangeably In the broadest sense and include monoclonal antibodies(e.g., full length or intact monoclonal antibodies), polyclonalantibodies, multivalent antibodies or multispecific antibodies (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity).

More particularly, such molecule consists in a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds. Each heavy chain is comprised of a heavy chainvariable region (or domain) (abbreviated herein as HCVR or VH) and aheavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (PR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.effector cells) and the first component (Clq) of the classicalcomplement system.

The heavy chains of immunoglobulins can be divided into three functionalregions; the Fd region, the hinge region, and the Fc region (fragmentcrystallizable). The Fd region comprises the VH and CH1 domains and, incombination with the light chain, forms Fab—the antigen-bindingfragment. The Fc fragment is responsible for the immunoglobulin,effector functions, which includes, for example, complement fixation andbinding to cognate Fc receptors of effector cells. The hinge region,found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexiblespacer that allows the Fab portion to move freely in space relative tothe Fc region. The hinge domains are structurally diverse, varying inboth sequence and length among immunoglobulin classes and subclasses.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided structurally and functionally into threeregions: the upper hinge, the core, and the lower hinge (Shin et al.,Immunological Reviews 130:87, 1992). The upper hinge includes aminoacids from the carboxyl end of CH1 to the first residue in the hingethat restricts motion, generally the first cysteine residue that formsan interchain disulfide bond between the two heavy chains. The length ofthe upper hinge region correlates with the segmental flexibility of theantibody. The core hinge region contains the inter-heavy chain disulfidebridges. The lower hinge region joins the amino terminal end of, andincludes residues in the CH2 domain. The core hinge region of human IgG1contains the sequence Cys-Pro-Pro-Cys that, when dimerized by disulfidebond formation, results in a cyclic octapeptide believed to act as apivot, thus conferring flexibility. Conformational changes permitted bythe structure and flexibility of the immunoglobulin hinge-regionpolypeptide sequence may affect the effector functions of the Fe portionof the antibody.

The term. <<Monoclonal Antibody>> is used in accordance with itsordinary meaning to denote an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. In otherwords, a monoclonal antibody consists in a homogenous antibody resultingfrom the proliferation of a single clone of cells (e.g., hybridomacells, eukaryotic host cells transfected with DNA encoding thehomogenous antibody, prokaryotic host cells transformed with DNAencoding the homogenous antibody, etc.), and which is generallycharacterized by heavy chains of a single class and subclass, and lightchains of a single type. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibodies preparations that typically include differentantibodies directed against different determinants, or epitope, eachmonoclonal antibody is directed against a single determinant on theantigen.

In the present description, the terms polypeptides, polypeptidesequences, amino acid sequences, peptides and proteins attached toantibody compounds or to their sequence are interchangeable.

The invention relates to a monoclonal antibody, or a divalent functionalfragment or derivative thereof, capable to inhibit the c-Metdimerization and comprising a heavy chain comprising CDR-H1, CDR-H2 andCDR-H3 with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3or a sequence having at least 80% identity after optimum alignment withsequences SEQ ID Nos. 1, 2 and 3; and a light chain comprising CDR-L1,CDR-L2 and CDR-L3 with respectively the amino acid sequences SEQ ID Nos.5, 6 and 7 or a sequence having at least 80% identity after optimumalignment with sequences SEQ ID Nos. 5, 6 or 7, said antibody beingfurther characterized in that it also comprises a hinge regioncomprising the amino acid sequence SEQ ID No. 56.

More particularly, the invention relates to a monoclonal antibody, or adivalent functional fragment or derivative thereof, as above describedcharacterized in that it also comprises a hinge region comprising theamino acid sequence SEQ ID No. 57.

In other words, the invention relates to a monoclonal antibody, or adivalent functional fragment or derivative thereof, capable to inhibitthe c-Met dimerization and comprising a heavy chain comprising CDR-H1,CDR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos.1, 2 and 3 or a sequence having at least 80% identity after optimumalignment with sequences SEQ ID Nos. 1, 2 and 3; and a light chaincomprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acidsequences SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80%identify after optimum alignment with sequences SEQ ID Nos. 5, 6 or 7,said antibody being further characterized in that it also comprises ahinge region comprising the amino acid sequence SEQ ID No. 5.

More particularly, the invention relates to a monoclonal antibody, or adivalent functional fragment or derivative thereof, as above describedcharacterized in that it also comprises a hinge region comprising theamino acid sequence SEQ ID No. 21.

In other words, the invention also relates to a monoclonal antibody, ora divalent functional fragment or derivative thereof capable to inhibitthe c-Met dimerization and comprising a heavy chain comprising CDR-H1,CPR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos.1, 2 and 3 or a sequence having at least 80% identity after optimumalignment with sequences SEQ ID Nos. 1, 2 and 3; and a light chaincomprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acidsequences SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80%identify after optimum alignment with sequences SEQ ID Nos. 5, 6 or 7,said antibody being further characterized in that it also comprises ahinge region comprising the amino acid sequence SEQ ID No. 21.

As it will be apparent for the man skilled in the art, the consensussequences SEQ ID Nos. 57 and 21 are comprised in the consensus sequenceSEQ ID No. 56.

TABLE 1 #01 #02 #03 #04 #05 #06 #07 #08 #09 #10 #11 #12 #13 #14 SEQ IDX1 X2 X3 C X5 X6 X7 X8 X9 C X11 X12 C X14 NO 56 SEQ ID X1 X2 X3 C X5 X6X7 X8 X9 C P P C P NO 57 SEQ ID X1 X2 X3 C X5 — C X8 X9 C X11 X12 C X14NO 21 For SEQ ID No. 56: X1: P, R, C, — X5: D, C, C, — X8: B, V, K, —X12: P, — X2: K, C, R, — X6: K, C, — X9: T, C, E, P, — X14: P, F X3: S,C, D, — X7: T, C, — X11: P, T

The expression “functional fragments and derivatives” will be defined indetails later in the present specification.

By CDR regions or CDR(s), it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulins as definedby IMGT.

The IMGT unique numbering has been defined to compare the variabledomains whatever the antigen receptor, the chain type, or the species[Lefranc M.-P., Immunology Today 18, 509 (1997); Lefranc M.-P., TheImmunologist, 7, 132-136 (1999); Lefranc, M.-P., Pommié, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT uniquenumbering, the conserved amino acids always have the same position, forinstance cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP),hydrophobic amino acid 89, cysteine 104 (2nd-CYS) phenylalanine ortryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT; 39 to 55, FR3-IMGT: 66 to 104 and: FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT; 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths (shown between brackets andseparated by dots, e.g. [8.8.13]) become crucial information. The IMGTunique numbering is used is 2D graphical representations, designated asIMGT Colliers de Perles [Ruiz, M, and Lefranc, M.-P., Immunogenetics,53, 857-882 (2002); Kaas, Q, and Lefranc, M.-P., Current Bioinformatics,2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Raas, Q.Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data,Nucl. Acids. Res., 32, D208-D210 (2004)].

Three heavy chain CDRs and 3 light chain CDRs exist. The term CDR orCDRs is used here in order to indicate, according to the case, one ofthese regions or several, or even the whole, of these regions whichcontain the majority of the amino acid residues responsible for thebinding by affinity of the antibody tor the antigen or the epitope whichit recognizes.

By “percentage of identity” between two nucleic acid or amino acidsequences in the sense of the present invention, it is intended toindicate a percentage of nucleotides or of identical amino acid residuesbetween the two sequences to be compared, obtained after the bestalignment (optimum alignment), this percentage being purely statisticaland the differences between the two sequences being distributed randomlyand over their entire length. The comparisons of sequences between twonucleic acid or amino acid sequences are traditionally carried out bycomparing these sequences after having aligned them in an optimummanner, said comparison being able to be carried out by segment or by“comparison window”. The optimum alignment of the sequences for thecomparison can be carried out, in addition to manually, by means of thelocal homology algorithm of Smith and Waterman (1981) [Ad. App. Math.2:482], by means of the local homology algorithm of Neddleman and Wunsch(1970) [J. Mol. Biol. 48:443], by means of the similarity search methodof Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), bymeans of computer software using these algorithms (GAP, BESTFIT, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis., or else by BLAST N or BLAST Pcomparison software).

The percentage of identity between two nucleic acid or amino acidsequences is determined by comparing these two sequences aligned in anoptimum manner and in which the nucleic acid or amino acid sequence tobe compared can comprise additions or deletions with respect to thereference sequence for an optimum alignment between these two sequences.The percentage of identity is calculated by determining the number ofidentical positions for which the nucleotide or the amino acid residueis identical between the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 in order to obtainthe percentage of identity between these two sequences.

For example, it is possible to use the BLAST program, “BLAST 2sequences” (Tatusova et al., “Blast 2 Sequences—a new tool for comparingprotein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250)available on the site http://www.ncbi.nlm.hih.gov/gorf/Bl2.html, theparameters used being those given by default (in particular for theparameters “open gap penalty”: 5, and “extension gap penalty”: 2; thematrix chosen being, for example, the matrix “BLOSUM 62” proposed by theprogram), the percentage of identity between the two sequences to becompared being calculated directly by the program.

By amino acid sequence having at least 80%, preferably 85%, 90%, 95% and98% identity with a reference amino acid sequence, those having, withrespect to the reference sequence, certain modifications, in particulara deletion, addition or substitution of at least one amino acid, atruncation or an elongation are preferred. In the case of a substitutionof one or more consecutive or nonconsecutive amino acid(s), thesubstitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. The expression “equivalent aminoacids” is aimed here at indicating any amino acid capable of beingsubstituted with one of the amino acids of the base structure without,however, essentially modifying the biological activities of thecorresponding antibodies and such as will be defined later, especiallyin the examples. These equivalent amino acids can be determined eitherby relying on their structural homology with the amino acids which theyreplace, or on results of comparative trials of biological activitybetween the different antibodies capable of being carried out.

By way of example, mention is made of the possibilities of substitutioncapable of being carried out without resulting in a profoundmodification of the biological activity of the corresponding modifiedantibody.

As non limitative example, the following table 2 is giving substitutionpossibilities conceivable with a conservation of the biological activityof the modified antibody. The reverse substitutions are also, of course,possible in the same conditions.

TABLE 2 Original residue Substitution(s) Ala (A) Val, Gly, Pro Arg (R)Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (G) Asp Gly(G) Ala His (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met(M) Leu Phe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) TyrTyr (Y) Phe, Trp Val (V) Leu, Ala

It must be understood here that the invention does not relate to theantibodies in natural form, that is to say they are not in their naturalenvironment but that they have been able to be isolated or obtained bypurification from natural sources, or else obtained by geneticrecombination, or by chemical synthesis, and that they can then containunnatural amino acids as will be described further on.

It must also be understood, as previously mentioned, that the inventionconcerns more particularly a chimeric and/or a humanized divalentantibody, or any divalent functional fragment or derivative, with anantagonistic activity. Divalent antibodies of the prior art are agonistsor partial agonists. The monoclonal antibody of the invention, includinga modified hinge as previously described, i.e. including a hinge regioncomprising the amino acid sequence SEQ ID No. 56, 57 or 21, is novel andpresents the particularity to have a improved antagonistic activitycompared to the chimeric or humanized antibody 224G11 without such amodified hinge as it will appear from the following examples.

Contrary to the prior art, inventors have obtained an improvedantagonistic activity without modifying the format of the antibody.Actually, in the closest prior art represented by the antibody 5D5, ithas been necessary to develop a monovalent fragment of the antibody togenerate an antagonistic activity. In the present application, by theuse of the hinge of the invention, it is possible for the first time toobtain a full divalent antibody with increased antagonistic activity,and this contrary to the general knowledge.

In a preferred embodiment, the antibody of the invention comprises ahinge region comprising an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 22 to 28 and 38 to 72, or a sequence having atleast 80% identity after optimum alignment with sequences SEQ ID Nos. 22to 28 and 58 to 72.

For more clarity, the following tables 3 and 4 regroup the amino acidsand nucleotides sequences of the different preferred hinges of theinvention.

TABLE 3 SEQ SEQ ID ID No. Amino acids No. Nucleotides 22 RKCCVECPPCP 29AGGAAGTGCTGTGTGGAGTGCCCCCCCTGCCCA 23 PRDCGCKPCICT 30CCCCGGGACTGTGGGTGCAAGCCTTGCATTTGTACC 24 PKSCGCKPCICT 31CCCAAGAGCTGTGGGTGCAAGCCTTGCATTTGTACC 25 PKSCGCKPCICP 32CCAAAGAGCTGCGGCTGCAAGCCTTGTATCTGTCCC 26 PRDCGCKPCPPCP 33CCACGGGACTGTGGCTGCAAGCCCTGCCCTCCGTGTCCA 27 PRDCGCHTCPPCP 34CCCAGAGACTGTGGGTGTCACACCTGCCCTCCTTGTCCT 28 PKSCDCHCPPCP 35CCCAAAAGCTGCGATTGCCACTGTCCTCCATGTCCA

TABLE 4 SEQ SEQ ID No. Amino acids ID No. Nucleotides 58 CKSCDKTHTCPPCP73 TGCAAGAGCTGCGACAAGACCCACACCTGTCCCCCCTGCCCT 59 PCSCDKTHTCPPCP 74CCCTGCAGCTGCGACAAGACCCACACCTGTCCCCCCTGCCCT 60 PKCCDKTHTCPPCP 75CCCAAGTGCTGCGACAAGACCCACACCTGTCCCCCCTGCCCT 61 PKSCCKTHTCPPCP 76CCTAAGAGCTGTTGCAAGACCCACACCTGTCCCCCCTGCCCT 62 PKSCDCTHTCPPCP 77CCCAAGAGCTGCGACTGCACCCACACCTGTCCCCCCTGCCCT 63 PKSCDKCHTCPPCP 78CCCAAGAGCTGCGACAAGTGCCACACCTGTCCCCCCTGCCCT 64 PKSCDKTHCCPPCP 79CCCAAGAGCTGCGACAAGACCCACTGCTGTCCCCCCTGCCCT 65 KCDKTHTCPPCP 80AAGTGCGACAAGACCCACACCTGTCCCCCCTGCCCT 66 PKSCDCHTCPPCP 81CCCAAGAGCTGCGACTGCCACACCTGTCCCCCCTGCCCT 67 PKSCDCTHCPPCP 82CCCAAGAGCTGCGACTGCACCCACTGCCCCCCCTGCCCT 68 PCSCKHTCPPCP 83CCCTGCAGCTGCAAGCACACCTGTCCCCCCTGCCCT 69 PSCCTHTCPPCP 84CCTAGCTGCTGCACCCACACCTGTCCCCCCTGCCCT 70 PSCDKHCCPPCP 85CCCAGCTGCGACAAGCACTGCTGCCCCCCCTGCCCT 71 PKSCTCPPCP 86CCCAAGAGCTGCACCTGTCCCCCTTGTCCT 72 PKSCDKCVECPPCP 87CCCAAGAGCTGCGATAAGTGCGTGGAGTGCCCCCCTTGTCCT

According a first approach, the antibody will be defined by its heavychain sequence. More particularly, the antibody of the invention, or oneof its functional fragments or derivatives, is characterized in that itcomprises a heavy chain comprising at least one CDR chosen from CDRscomprising the amino acid sequences SEQ ID Nos. 1 to 3.

The mentioned sequences are the following ones:

SEQ ID No. 1: GYIFTAYT SEQ ID No. 2: IKPNNGLA SEQ ID No. 3: ARSEITTEFDY

According to a preferred aspect, the antibody of the invention, or oneof its functional fragments or derivatives, comprises a heavy chaincomprising at least one, preferably two, and most preferably three,CDR(s) chosen from CDR-H1, CDR-H2 and CDR-H3, wherein:

-   -   CDR-H1 comprises the amino acid sequence SEQ ID No. 1,    -   CDR-H2 comprises the amino acid sequence SEQ ID No. 2,    -   CDR-H3 comprises the amino acid sequence SEQ ID No. 3.

In a second approach, the antibody will be now defined by its lightchain sequence. More particularly, according to a second particularaspect of the invention, the antibody, or one of its functionalfragments or derivatives, is characterized in that it comprises a lightchain comprising at least one CDR chosen from CDRs comprising the aminoacid sequence SEQ ID Nos. 5 to 7.

The mentioned sequences are the following ones:

SEQ ID No. 5: ESVDSYANSF SEQ ID No. 6: RAS SEQ ID No. 7: QQSKEDPLT

According to another preferred aspect, the antibody of the invention, orone of its functional fragments or derivatives, comprises a light chaincomprising at least one, preferably two, and most preferably three,CDR(s) chosen, from CDR-L1, CDR-L2 and CDR-L3, wherein:

-   -   CDR-L1 comprises the amino acid sequence SEQ ID No. 5,    -   CDR-L2 comprises the amino acid sequence SEQ ID No. 6,    -   CDR-L3 comprises the amino acid sequence SEQ ID No. 7.

The murine hybridoma capable of secreting monoclonal antibodiesaccording to the present invention, especially hybridoma of murineorigin, was deposited at the CNCM (Institut Pasteur, Paris, France) onMar. 14, 2007 under the number CNCM I3731.

In the present application, IgG1 are preferred to get effectorfunctions, and most preferably ADCC and CDC.

The skilled artisan will recognize that effector functions include, forexample, Clq binding; complement dependent cytotoxicity (CDC); Fcreceptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; and down regulation of cell surface receptors (e.g. B cellreceptor; BCR).

The antibodies according to the present invention, are preferablyspecific monoclonal antibodies, especially of murine, chimeric orhumanized origin, which can be obtained according to the standardmethods well known to the person skilled in the art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments or derivatives, especially of murine origin, it ispossible to refer to techniques which are described in particular in themanual “Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp, 726, 1988)or to the technique of preparation from hybridomas described by Kohlerand Milstein (Nature, 256:495-49, 1975).

The monoclonal antibodies according to the invention can be obtained,for example, from an animal cell immunized against the c-Met, or one ofits fragments containing the epitope specifically recognized by saidmonoclonal antibodies according to the invention. Said c-Met, or one ofits said fragments, can especially be produced according to the usualworking methods, by genetic recombination starting with a nucleic acidsequence contained in the CDNA sequence coding for the c-Met or bypeptide synthesis starting from a sequence of amino acids comprised inthe peptide sequence of the c-Met.

The monoclonal antibodies according to the invention can, for example,be purified on an affinity column on which the c-Met or one of itsfragments containing the epitope specifically recognized by saidmonoclonal antibodies according to the invention has previously beenimmobilized. More particularly, said monoclonal antibodies can bepurified by chromatography on protein A and/or G, followed or notfollowed by ion-exchange chromatography aimed at eliminating theresidual protein contaminants as well as the DNA and the LPS, in itselffollowed or not followed by exclusion chromatography on Sepharose™ gelin order to eliminate the potential aggregates due to the presence ofdimers or of other multimers. In an even more preferred manner, thewhole of these techniques can be used simultaneously or successively.

The antibody of the invention, or a divalent functional fragment orderivative thereof, consists preferably of a chimeric antibody.

By chimeric antibody, it is intended to indicate an antibody whichcontains a natural variable (light chain and heavy chain) region derivedfrom an antibody of a given species in combination with the light chain,and heavy chain constant regions of an antibody of a speciesheterologous to said given species (e.g. mouse, horse, rabbit, dog, cow,chicken, etc.).

The antibodies or their fragments of chimeric type according to theinvention can be prepared by using the techniques of geneticrecombination. For example, the chimeric antibody can be produced bycloning a recombinant DNA containing a promoter and a sequence codingfor the variable region of a non-human, especially murine, monoclonalantibody according to the invention and a sequence coding for theconstant region of human antibody. A chimeric antibody of the inventionencoded by such a recombinant gene will be, for example, a mouse-manchimera, the specificity of this antibody being determined by thevariable region derived from the murine DNA and its isotype determinedby the constant region derived from the human DNA. For the methods ofpreparation of chimeric antibodies, it is possible, for example, torefer to the documents Verhoeyn et al. (BioEssays, 8:74, 1988), Morrisonet al. (Proc. Natl. Acad. Sci. USA 82:6851-6855, 1984) or U.S. Pat. No.4,816,567.

More particularly, said antibody, or a functional fragment or derivativethereof comprises a chimeric heavy chain variable domain of sequencecomprising the amino acid sequence SEQ ID No. 46 or a sequence having atleast 80% identity after optimum alignment with the sequence SEQ ID No.46.

SEQ ID No. 46: EVQLQQSGPELVKPGASVKISCKTSGYIFTAYTMHWVRQSLGESLDWIGGIKPNNGLANYNQKFKGKATLTVDKSSSSTAYMDLRSLTSEDSAVYYCARSEITTEFDYWGQGTALTVSS

More particularly, said antibody, or a functional fragment or derivativethereof, comprises a chimeric light chain variable domain of sequencecomprising the amino acid sequence SEQ ID No. 47 or a sequence having atleast 80% identity after optimum alignment with the sequence SEQ ID No.47.

SEQ ID No. 47: DIVLTQSPASLAVSLGQRATISCRASESVDSYANSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSKEDP LTFGSGTKLEMKR

More particularly, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention, and named[224G11] [IgG2chim], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a tight chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 22.

In the present application, the use of square brackets is not necessaryand, as an example, the reference [224G11] [IgG2chim] must be consideredas identical to 224G11IgG2chim. In a same way, to indicate that theantibody is a murine one, the expression murine or the letter m can beadded; to indicate that the antibody is a chimeric one, the expressionchim or the letter c can be added and; to indicate that the antibody isa humanized one, the expression hum, hx, Hz or the letter h can beadded. As an example, the chimeric antibody 224G11gG2 can be referred asc224G11IgG2, c224G11[IgG2], c[224G11]IgG2, c[224G11][IgG2],224G11IgG2chim, 224G11[IgG2chim], [224G11]IgG2chim or[224G11][IgG2chim].

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [TH7chim], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a tight chain variable domaincomprising the amino acid sequence SEQ. ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 28.

In the present application, the reference TH7 must be considered asidentical to C7Δ6-9 or TH7C7Δ6-9. The symbol Δ means deletion.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [MHchim], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a light chain, variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 23.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [MUP9Hchim], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 26.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [MMCHchim], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 24.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [C2], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain, variable domain,comprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 58.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [C2], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 59.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [C3], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 60.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [C5], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain, variable domain,comprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 61.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [C6], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 62.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [C7], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain, variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 63.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [C9], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 64.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [Δ1-3], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain variable domain,comprising the amino acid sequence SEQ ID No. 47, and a binge regioncomprising the amino acid sequence SEQ ID No. 65.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [C7Δ6], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 66.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [C6Δ9], comprises a heavy chain variable domain comprising theamino acid sequence SEQ ID No. 46, a light chain, variable domain,comprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 67.

In another aspect, a preferred antibody, or a divalent; functionalfragment or derivative thereof according to the-invention and named[224G11] [C2Δ5-7], comprises a heavy chain, variable domain comprisingthe amino acid Sequence SEQ ID No. 46, a light chain variable domain,comprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 68.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention, and named[224G11] [C5Δ2-6], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 69.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [C9Δ2-7], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 70.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [Δ5-6-7-8], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 46, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 47, and a hinge regioncomprising the amino acid sequence SEQ ID No. 71.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11] [IgG1/IgG2], comprises a heavy chain variable domaincomprising, the amino acid sequence SEQ ID No. 46, a light chain,variable domain comprising the amino acid sequence SEQ ID No. 47, and ahinge region comprising the amino acid sequence SEQ ID No. 72.

The antibody of the invention, or a divalent Emotional fragment orderivative thereof consists preferably of a human antibody.

The term “human antibody” includes all antibodies that have one or morevariable and constant region, derived from human immunoglobulinsequences. In a preferred embodiment, all of the variable and constantdomains (or regions) are derived from human immunoglobulin sequence(fully human antibody). In other words, it includes any antibody whichhave variable and constant regions (if present) derived from humangermline immunoglobulin sequences, i.e. which possesses an amino acidsequence which corresponds to that of an antibody produced by a humanand/or has been made using any techniques for making-human antibodiesknown by the man skill in the art.

In one embodiment, the human monoclonal antibodies arc produced by ahybridoma which includes a B cell obtained from a transgenic non-humananimal, e.g., a transgenic mouse, having a genome comprising a humanheavy chain transgene and a light chairs transgene fused to animmortalized cell.

As example for such transgenic mouse, it can be mentioned the XENOMOUSE™which is an engineered mouse strain that comprises large fragments ofthe human immunoglobulin loci and is deficient in mouse antibodyproduction (Green et al., 1994, Nature Genetics, 7: 13-21). TheXENOMOUSE™ produces an adult-like human repertoire of fully humanantibodies, and generate antigen-specific human monoclonal antibodies. Asecond generation XENOMOUSE™ contains approximately 80% of the humanantibody repertoire (Green & Jakobovits, 1998, J. Exp. Med.,188:483-495).

Any other technique known, by the man skill in the art, such as phagedisplay technique, can also be used for the generation of human antibodyaccording to the invention.

The antibody of the invention, or a divalent functional fragment orderivative thereof, consists preferably of a humanized antibody.

By the expression “humanized antibody”, it is intended to indicate anantibody which contains CDR regions derived from an antibody of nonhumanorigin, the other parts of the antibody molecule being derived from one(or from several) human antibodies. Moreover, some of the residues ofthe segments of the skeleton (called FR) can be modified in order toconserve the affinity of the binding (Jones et al., Nature, 321:522-525,1986; Verhoeyen et al., Science, 239:1524-1536, 1988; Riechmann et al.Nature, 332:323-327, 1988).

The humanized antibodies according to the invention or their fragmentscan be prepared by techniques known to the person skilled in the art(such as, for example, those described in the documents Singer et al.,J. Immun. 150:2844-2857, 1992; Mountain et al. Biotechnol. Genet. Eng.Rev., 10: 1-142, 1992; or Bebbington et al., Bio/Technology, 10:169-175,1992).

Other humanization method are known by the man skill in the art as, forexample, the “CDR Grafting” method described by Protein Design Lab (PDL)in the patent applications EP 0 451261, EP 0 682 040, EP 0 9127, EP 0566 647 or U.S. Pat. No. 5,530,101, U.S. Pat. No. 6,180,370, U.S. Pat.No. 5,585,089 and U.S. Pat. No. 5,693,761. The following patentapplications can also be mentioned: U.S. Pat. No. 5,639,641; U.S. Pat.No. 6,054,297; U.S. Pat. No. 5,886,152 and U.S. Pat. No. 5,877,293.

More particularly, said antibody, or a functional fragment or derivativethereof, comprises a humanized heavy chain variable domain of sequencecomprising the amino acid sequence SEQ ID No. 4 or a sequence having atleast 80% identity after optimum alignment with the sequence SEQ ID No.4.

SEQ ID No. 4: QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPGQGLEWMGWIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSEITTEFDYWGQGTLVTVSS

More particularly, said antibody, or a functional fragment or derivativethereof, comprises a humanized light chain, variable domain selectedfrom the group of sequences comprising the amino acid sequence SEQ IDNo. 8, 9 or 10 or a sequence having at least 80% identify after optimumalignment with the sequence SEQ ID No. 8, 9 or 10.

SEQ ID No. 8: DIVLTQSPDSLAVSLGERATINCKSSESVDSYANSFMHWYQQKPGQPPKLLIYRASTRESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSKEDP LTFGGGTKVEIKRSEQ ID No. 9: DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFMHWYQQKPGQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDP LTFGGGTKVEIKRSEQ ID No. 10: DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKPGQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYVQQSKEDP LTFGGGTKVEIKR

More particularly, a preferred antibody, or a divalent functionalfragment or derivative thereof, according to the invention and named[224G11]60 [IgG2Hz1], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 4, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 8, and a fringe regioncomprising the amino acid sequence SEQ ID No. 22.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [IgG2Hz2], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 4, a light chain variable domaincomprising die amino acid sequence SEQ ID No. 9, and a hinge regioncomprising the ammo acid sequence SEQ ID No. 22.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention, and named[224G11] [IgG2Hz3], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 4, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 10, and a hinge regioncomprising the amino acid sequence SEQ ID No. 22.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the Invention and named[224G11] [TH/Hz1], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 4, a light chain, variable domaincomprising the amino acid sequence SEQ ID No. 8, and a hinge regioncomprising the amino acid sequence SEQ ID No. 28.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [TH7Hz2], comprises a heavy chains variable: domain comprisingthe amino acid sequence SEQ ID No. 4, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 9, and a hinge regioncomprising the amino acid sequence SEQ ID No. 28.

In another aspect, a preferred antibody, or a divalent functionalfragment or derivative thereof according to the invention and named[224G11] [TH7Hz3], comprises a heavy chain variable domain comprisingthe amino acid sequence SEQ ID No. 4, a light chain variable domaincomprising the amino acid sequence SEQ ID No. 10, and a hinge regioncomprising the amino acid sequence SEQ ID No. 28.

In another aspect, antibodies of the invention can be described by theirtotal heavy and light chains, respectively.

As example, the antibody [224G1] [IgG2chim] of the invention comprises acomplete heavy chain comprising the amino acid sequence SEQ ID No. 50,or a sequence having at least 80% identity after optimum alignment withthe sequence SEQ ID No. 50, and a complete light chain comprising theamino acid sequence SEQ ID No. 52, or a sequence having at least 80%identity after optimum alignment with the sequence SEQ ID No. 52.

As another example, the antibody [224G11] [TH7chim] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 51, or a sequence having at least 80%, identity after optimumalignment with the sequence SEQ ID No. 51, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C1] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 88, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 88, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C2] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 89, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 89, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C3] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 90, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 90, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C5] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 91, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 91, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C6] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 92, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 92, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C7] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 93, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 93, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C9] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 94, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 94, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [Δ1-3] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 95, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 95, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C7Δ6] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 96, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 96, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C6Δ9] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 97, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 97, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C2Δ5-7] of the invention,comprises a complete heavy chain comprising the amino acid sequence SEQID No. 98, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 98, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C5Δ2-6] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 99, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 99, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [C9Δ2-7] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 100, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 100, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [Δ5-6-7-8] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 101, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 101, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [IgG1/IgG2] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 102, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 102, and a complete light chaincomprising the amino acid sequence SEQ ID No. 52, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 52.

As another example, the antibody [224G11] [IgG2Hz1] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 36, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 36 and a complete light chaincomprising the amino acid sequence SEQ ID No. 38, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 38.

As another example, the antibody [224G11] [IgG2Hz2] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 36, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 36 and a complete light chaincomprising the amino acid sequence SEQ ID No. 39, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 39.

As another example, the antibody [224G11] [IgG2Hz3] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 36, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 36 and a complete light chaincomprising the amino acid sequence SEQ ID No. 40, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 40.

As another example, the antibody [224G11] [TH7Hz1] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 37, or a sequence having at least 80% identity after optimismalignment with the sequence SEQ ID No. 37 and a complete light chaincomprising the amino acid sequence SEQ ID No. 38, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 38.

As another example, the antibody [224G11] [TH7Hz2] of the inventioncomprises a complete heavy chain comprising die amino acid sequence SEQID No. 37, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 37 and a complete light chaincomprising the amino acid sequence SEQ ID No. 39, or a sequence havingat least 80% identify alter optimum alignment with the sequence SEQ IDNo. 39.

As another example, the antibody [224G11] [TH7Hz3] of the inventioncomprises a complete heavy chain comprising the amino acid sequence SEQID No. 37, or a sequence having at least 80% identity after optimumalignment with the sequence SEQ ID No. 37 and a complete light chaincomprising the amino acid sequence SEQ ID No. 40, or a sequence havingat least 80% identity after optimum alignment with the sequence SEQ IDNo. 40.

Other examples of antibodies, or derivatives thereof, according to theinvention comprises complete heavy chains comprising an amino acidsequence selected in the group consisting of SEQ ID Nos. 88 to 102(corresponding nucleotide sequences are SEQ ID Nos. 103 to 117).

By “functional fragment” of an antibody according to the invention, itis intended to indicate in particular an antibody fragment, such as Fv,scFv (sc for single chain), Fab, F(ab′)₂, Fab′, scFv-Fc fragments ordiabodies, or any fragment of which the half-life time would have beenincreased by chemical modification, such as the addition ofpoly(alkylene) glycol such as poly(ethylene) glycol (“PEGylation”)(pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂-PEG orFab′-PEG) (“PEG” for Poly(Ethylene) Glycol), or by incorporation in aliposome, said fragments having at least one of the characteristic CDRsof sequence SEQ ID Nos. 1 to 3 and 5 to 7 according to the invention,and, especially, in that it is capable of exerting in a general manneran even partial activity of the antibody from which it is descended,such as in particular the capacity to recognize and to bind to thec-Met, and, if necessary, to inhibit the activity of the c-Met.

Preferably, said functional fragments will be constituted or willcomprise a partial sequence of the heavy or light variable chain of theantibody from which they are derived, said partial sequence beingsufficient to retain the same specificity of binding as the antibodyfrom which it is descended and a sufficient affinity, preferably atleast equal to 1/100, in a more preferred manner to at least 1/10, ofthat of the antibody from which it is descended, with respect to thec-Met. Such a functional fragment will contain at the minimum 5 aminoacids, preferably 6, 7, 8, 9, 10, 12, 15, 25, 50 and 100 consecutiveamino acids of the sequence of the antibody from which it is descended.

Preferably, these functional fragments will be fragments of Fv, scFv,Fab, F(ab′)₂, F(ab′), scFv-Fc type or diabodies, which generally havethe same specificity of binding as the antibody from which they aredescended. In a more preferred embodiment of the invention, thesefragments are selected among divalent fragments such as F(ab′)₂fragments. According to the present invention, antibody fragments of theinvention can be obtained starting from antibodies such as describedabove by methods such as digestion by enzymes, such as pepsin or papainand/or by cleavage of the disulfide bridges by chemical reduction. Inanother manner, the antibody fragments comprised in the presentinvention can be obtained by techniques of genetic recombinationlikewise well known to the person skilled in the art or else by peptidesynthesis by means of, for example, automatic peptide synthesizers suchas those supplied by the company Applied Biosystems, etc.

By “divalent fragment”, it must be understood any antibody fragmentscomprising two arms and, more particularly, F(ab′)₂ fragments.

By “derivatives” of an antibody according to the invention, it is meanta binding protein comprising a protein scaffold and at least on of theCDRs selected from the original antibody in order to maintain, thebinding capacity. Such compounds are well known by the man skilled inthe art and will be described in more details in the followingspecification.

More particularly, the antibody, or one of its functional fragments orderivatives, according to the invention is characterized in that saidderivative consists in a binding protein comprising a scaffold on whichat least one GDR has been grafted for the conservation of the originalantibody paratopic recognizing properties.

One or several sequences through the 6 GDR sequences described in theinvention can be presented on a protein scaffold. In this case, theprotein scaffold reproduces the protein backbone with appropriatefolding of the grafted CDR(s), thus allowing it (or them) to maintaintheir antigen paratopic recognizing properties.

The man skilled in the art knows how to select the protein scaffold onwhich at least one CDR selected from the original antibody could begrafted. More particularly, it is known that, to be selected, suchscaffold should display several features as follow (Skerra A., J. Mol.Recogn., 13, 2000, 167-187):

-   -   phylogenetically good conservation,    -   robust architecture with a well known three-dimensional        molecular organization (such as, for example, crystallography or        NMR),    -   small size,    -   no or only low degree of post-translational modifications,    -   easy to produce, express and purify.

Such protein scaffold can be, but without limitation, structure selectedfrom the group consisting in fibronectin and preferentially the tenthfibronectin type III domain (FNfn10), lipocalin, anticalin (Skerra A.,J. Biotechnol., 2001, 74(4):257-75), the protein Z derivative from thedomain B of staphylococcal protein A, thioredoxin A or any protein withrepeated domain such as “armadillo repeat” (Kohl et al., PNAS, 2003,vol. 100, No.4, 1700-1705), “armadillo repeat ” “leucin-rich repeat” or“tetratricopeptide repeat”.

It could also be mentioned scaffold derivative from toxins (such as, forexample, scorpion, insect, plant or mollusc toxins) or proteininhibitors of neuronal nitric oxyde synthase (PIN).

As non limitative example of such hybrid constructions, it can bementioned the insertion of the CDR-H1 (heavy chain) of an anti-CD4antibody, i.e. the 13B8.2 antibody, into one of the exposed loop of thePIN. The binding properties of the obtained binding protein remainsimilar to the original antibody (Bes et al., BBRC 343, 2006, 334-344).It can also be mentioned the grafting of the CDR-H3 (heavy chain) of ananti-lyzozyme VHH antibody on a loop of neocarzinostatine (Nicaise etal., 2004).

As above mentioned, such protein scaffold can comprise from 1 to 6CDR(s) from the original antibody. In a preferred embodiment, butwithout any limitation, the man skilled in the art would select at leasta CDR from the heavy chain, said heavy chain being known to beparticularly implicated in the antibody specificity. The selection ofthe CDR(s) of interest will be evident for the man of the art with knownmethod (BES et al., FEBS letters 508, 2001, 67-74).

As an evidence, these examples are not limitative and any other scaffoldknown or described must be included in the present specification.

According to a novel aspect, the present invention relates to anisolated nucleic acid, characterized in that it is chosen from thefollowing nucleic acids;

-   -   a) a nucleic acid, DNA or RNA, coding for an antibody, or one of        its functional fragments or derivatives, according to the        invention;    -   b) a nucleic acid sequence comprising the sequences SEQ ID No.        11, SEQ ID No. 12, SEQ ID No. 13 and the sequences SEQ ID No.        15, SEQ ID No. 16 and SEQ ID No. 17;    -   c) a nucleic acid sequence comprising the sequences SEQ ID No.        14 and SEQ ID No. 18, 19 or 20;    -   d) the corresponding RNA nucleic acids of the nucleic acids as        defined in b) or c);    -   e) the complementary nucleic acids of the nucleic acids as        defined in a), b) and c); and    -   f) a nucleic acid of at least 18 nucleotides capable of        hybridizing under conditions of high stringency with at least        one of the CDRs of sequence SEQ ID Nos. 11 to 13 and 15 to 17.

According to still another aspect, the present invention relates to asisolated nucleic acid, characterized in that it is chosen from thefollowing nucleic acids:

-   -   a nucleic acid, DNA or RNA, coding for an antibody, or one of        its functional fragments or derivatives, according to the        present invention and wherein the nucleic sequence coding for        the hinge region of said antibody comprises or has a sequence        selected from the group consisting of the sequences SEQ ID Nos.        29 to 35 and SEQ ID Nos. 73 to 87.

By nucleic acid, nucleic or nucleic acid sequence, polynucleotide,oligonucleotide, polynucleotide sequence, nucleotide sequence, termswhich will be employed indifferently in the present invention, it isintended to indicate a precise linkage of nucleotides, which aremodified or unmodified, allowing a fragment or a region of a nucleicacid to be defined, containing or not containing unnatural nucleotides,and being able to correspond just as well to a double-stranded DNA, asingle-stranded DNA as to the transcription products of said DNAs.

It must also be understood here that the present invention does notconcern the nucleotide sequences in their natural chromosomalenvironment, that is to say in the natural state. It concerns sequenceswhich have been isolated and/or purified, that is to say that they havebeen selected directly or indirectly, for example by copy, theirenvironment having been at least partially modified. If is thus likewiseintended to indicate here the isolated nucleic acids obtained by geneticrecombination by means, for example, of host cells or obtained bychemical synthesis.

A hybridization under conditions (if high stringency signifies that thetemperature conditions and ionic strength conditions are chosen in sucha way that they allow the maintenance of the hybridization between twofragments of complementary DNA. By way of illustration, conditions ofhigh stringency of the hybridization step for the purposes of definingthe polynucleotide fragments described above are advantageously thefollowing.

The DNA-DNA or DNA-RNA hybridization, is carried out in two steps: (1)prehybridizatlon at 42° C. for 3 hours in phosphate buffer (20 mM, pH7.5) containing 5 ×SSC (1×SSC corresponds to a 0.15 M NaCl+0.015 Msodium citrate solution), 50% of formamide, 7% of sodium dodecyl sulfate(SDS), 10×Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA;(2) actual hybridization for 20 hours at a temperature dependent on thesize of the probe (i.e.; 42° C., for a probe size >100 nucleotides)followed by 2 washes of 20 minutes at 20° C. in 2×SSC+2% of SDS, 1 washof 20 minutes at 20° C. in 0.1×SSC+0.1% of SDS. The last wash is carriedout in 0.1×SSC+0.1% of SDS for 30 minutes at 60° C. for a probesize >100 nucleotides. The hybridization conditions of high stringencydescribed above for a polynucleotide of defined size can be adapted bythe person skilled in the art for oligonucleotides of greater or smallersize, according to the teaching of Sambrook et al, (1989, Molecularcloning: a laboratory manual. 2nd Ed. Cold Spring, Harbor).

The invention likewise relates to a vector comprising a nucleic acidaccording to the present invention.

The invention aims especially at cloning and/or expression vectors whichcontain a nucleotide sequence according to tire invention.

The vectors according to the invention preferably contain elements whichallow the expression and/or the secretion of the translated nucleotidesequences in a determined host cell. The vector must therefore contain apromoter, signals of initiation and termination of translation, as wellas appropriate regions of regulation of transcription, it must be ableto be maintained in a stable manner in the host cell and can optionallyhave particular signals which specify the secretion of the translatedprotein. These different elements are chosen and optimized by the personskilled in the art as a function of the host cell used. To this effect,the nucleotide sequences according to the invention can be inserted intoautonomous replication vectors in the chosen host or be integrativevectors of the chosen host.

Such vectors are prepared by methods currently used by the personskilled in the art, and the resulting clones can be introduced into anappropriate host by standard methods, such as lipofection,electroporation, thermal shock, or chemical methods.

The vectors according to the invention are, for example, vectors ofplasmidic or viral origin. They are useful for transforming host cellsin order to clone or to express the nucleotide sequences according tothe invention.

The invention likewise comprises the host cells transformed by orcomprising a vector according to the invention.

The host cell can be chosen from prokaryotic or eukaryotic systems, forexample bacterial cells but likewise yeast cells or animal cells, inparticular mammalian cells. It is likewise possible to use insect cellsor plant cells.

The invention likewise concerns animals, except man, which comprise atleast one cell transformed according to the invention.

According to another aspect, a subject of the invention is a process forproduction of an antibody, or one of its functional fragments accordingto the invention, characterized in that it comprises the followingstages:

-   -   a) culture in a medium and appropriate culture conditions of a        host cell according to the invention; and    -   b) the recovery of said antibodies, or one of their functional        fragments, thus produced starting from the culture medium or        said cultured cells.

The cells transformed according to the invention can be used inprocesses for preparation of recombinant polypeptides according to theinvention. The processes for preparation of a polypeptide according tothe invention in recombinant form, characterized in that they employ avector and/or a cell transformed by a vector according to the invention,are themselves comprised in the present invention. Preferably, a celltransformed by a vector according to the invention is cultured underconditions which allow the expression of said polypeptide and saidrecombinant peptide is recovered.

As has been said, the host cell can be chosen from prokaryotic oreukaryotic systems. In particular, it is possible to identify nucleotidesequences according to the invention, facilitating secretion in such aprokaryotic or eukaryotic system. A vector according to the inventioncarrying such a sequence can therefore advantageously be used for theproduction of recombinant proteins, intended to be secreted. In effect,the purification of these recombinant proteins of interest will befacilitated by the fact that they are present in the supernatant of thecell culture rather than in the interior of the host cells.

It is likewise possible to prepare the polypeptides according to theinvention by chemical synthesis. Such a preparation process is likewisea subject of the invention. The person skilled in the art knows theprocesses of chemical synthesis, for example the techniques employingsolid phases [Steward et al., 1984, Solid phase peptide synthesis,Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)] or techniquesusing partial solid phases, by condensation of fragments or by aclassical synthesis in solution. The polypeptides obtained by chemicalsynthesis and being able to contain corresponding unnatural amino acidsare likewise comprised in the invention.

The antibodies, or one of their functional fragments or derivatives,capable of being obtained by a process according to the invention arelikewise comprised in the present invention.

The invention also concerns the antibody of the invention as amedicament.

The invention likewise concerns a pharmaceutical composition comprisingby way of active principle a compound consisting of an antibody, or oneof its functional fragments according to the invention, preferably mixedwith an excipient and/or a pharmaceutically acceptable vehicle.

Another complementary embodiment of the invention consists in acomposition such as described above which comprises, moreover, as acombination product for simultaneous, separate or sequential use, ananti-tumoral antibody.

Most preferably, said second anti-tumoral antibody could be chosenthrough anti-IGF-IR, anti-EGFR, anti-HER2/neu, anti-VEGFR, anti-VEGF,etc., antibodies or any other anti-tumoral antibodies known by the manskilled in the art. It is evident that the use, as second antibody, offunctional fragments or derivatives of above mentioned antibodies ispart of the invention.

As a most preferred antibody, anti-EGFR antibodies are selected such asfor example the antibody C225 (Erbitux).

“Simultaneous use” is understood as meaning the administration of thetwo compounds of the composition according to the invention to a singleand identical pharmaceutical form.

“Separate use” is understood as meaning the administration, at the sametime, of the two compounds of the composition according to the inventionin distinct pharmaceutical forms.

“Sequential use” is understood as meaning the successive administrationof the two compounds of the composition according to the invention, eachin a distinct pharmaceutical form.

In a general fashion, the composition according to the inventionconsiderably increases the efficacy of the treatment of cancer. In otherwords, the therapeutic effect of the anti-c-Met antibodies according tothe invention is potentiated in an unexpected manner by theadministration of a cytotoxic agent. Another major subsequent advantageproduced by a composition according to the invention concerns thepossibility of using lower efficacious doses of active principle, whichallows the risks of appearance of secondary effects to be avoided or tobe reduced, in particular the effects of the cytotoxic agent.

In addition, this composition according to the invention would allow theexpected therapeutic effect to be attained more rapidly.

The composition of the invention can also be characterized in that itcomprises, moreover, as a combination product for simultaneous, separateor sequential use, a cytotoxic/cytostatic agent.

By “anti-cancer therapeutic agents”or “cytotoxic/cytostatic agents” itis intended a substance which, when administered to a subject, treats orprevents the development of cancer in the subject's body. As nonlimitative example of such agents, it can be mentioned alkylatingagents, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors,chromatin function inhibitors, anti-angiogenesis agents, anti-estrogens,anti-androgens or immunomodulators.

Such agents are, for example, cited in the 2001 edition of VIDAL, on thepage devoted to the compounds attached to the cancerology and hematologycolumn “Cytotoxics”, these cytotoxic compounds cited with reference tothis document are cited here as preferred cytotoxic agents.

More particularly, the following agents are preferred according to theinvention.

“Alkylating agent” refers to any substance which can cross-link oralkylate any molecule, preferably nucleic acid (e.g., DNA) within acell. Examples of alkylating agents include nitrogen mustard such asmechlorethamine, chlorambucol, melphalen, chlorydrate, pipobromen,prednimustin, disodic-phosphate or estramustine; oxazophorins such ascyclophosphamide, altretamine, trofosfamide, sulfofosfamide orifosfamide; aziridines or imine-ethylenes such as thiotepa,triethylenamine or altetramine; nitrosourea such as carmustine,streptozocin, fotemustin or lomustine; alkyle-sulfonates such asfusulfan, treosulfan or improsulfan; triazenes such as decarbazine; orplatinum complexes such as cis-platinum, oxaliplatin and carboplatin.

“Anti-metabolites” refer to substances that block cell growth and/ormetabolism by interfering with certain activities, usually DNAsynthesis. Examples of anti-metabolites include methotrexate,5-fluoruracil, floxuridine, 5-fluorodeoxyuridine, capecitabine,cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine (6-MP),6-thioguanine (6-TG), chlorodesoxyadenosine, 5-azacytidine, gemcitabine,cladribine, deoxycoformycin and pentostatin.

“Anti-tumor antibiotics” refer to compounds which may prevent or inhibitDNA, RNA and/or protein synthesis. Examples of anti-tumor antibioticsinclude doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone,dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, andprocarbazine.

“Mitotic inhibitors” prevent normal progression, of the cell cycle andmitosis. In general microtubule inhibitors or taxoides such aspaclitaxel and docetaxel are capable of inhibiting mitosis. Vincaalkaloid such as vinblastine, vincristine, vindesine and vinorelnine arealso capable of inhibiting mitosis.

“Chromatin function inhibitors” or “topoisomerase inhibitors” refer tosubstances which inhibit the normal function of chromatin modelingproteins such as topoisomerase I or topoisomerase II. Examples ofchromatin function inhibitors include, for topoisomerase I,camptothecine and its derivatives such as topotecan or irinotecan, and,for topoisomerase II, etoposide, etoposide phosphate and teniposide.

“Anti-angiogenesis agent” refers to any drug, compound, substance oragent which inhibits growth of blood vessels. Exemplaryanti-angiogenesis agents include, but are by no means limited to,razoxin, marimastat, batimastat, prinomastat, tanomastat, ilomastat,CGS-27023A, halofuginon, COL-3, neovastat, BMS-275291, thalidomide, CDC501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668,interferon-alpha, EMD121974, interleukin-12, IM862, angiostatin andvitaxin.

“Anti-estrogen” or “anti-estrogenic agent” refer to any substance whichreduces, antagonizes or inhibits the action of estrogen. Examples ofanti-estrogen agents are tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, anastrozole, letrozole, and exemestane.

“Anti-androgens” or “anti-androgen agents” refer to any substance whichreduces, antagonizes or inhibits the action of an androgen. Examples ofanti-androgens are flutamide, nilutamide, bicalutamide, spironolactone,cyproterone acetate, finasteride and cimitidine.

“Immunomodulators” are substances which stimulate the immune system.

Examples of immunomodulators include interferon, interleukin such asaldesleukine, OCT-43, denileukin diflitox and interleukin-2, tumoralnecrose fators such as tasomermine or others immunomodulators such aslentinan, sizofiran, roquinimex, pidotimod, pegademase, thymopentine,poly I:C or levamisole in conjunction with 5-fluorouracil.

For more detail, the man skill in the art could refer to the manualedited by the “Association Française des Enseignants de ChimieThérapeutique” and entitled “Traité de chimie thérapeutique”, vol. 6,Médicaments antitumoraux et perspectives dans le traitement des cancers,edition TEC & DOC, 2003.

Can also be mentioned as chemical agents or cytotoxic agents, all kinaseinhibitors such as, for example, gefitinib or erlotinib.

In a particularly preferred embodiment, said composition as acombination product according to the invention is characterized in thatsaid cytotoxic agent is coupled chemically to said antibody forsimultaneous use.

In order to facilitate the coupling between said cytotoxic agent andsaid antibody according to the invention, it is especially possible tointroduce spacer molecules between the two compounds to be coupled, suchas poly(alkylene) glycols like polyethylene glycol, or else amino acids,or, in another embodiment, to use active derivatives of said cytotoxicagents into which would have been introduced functions capable ofreacting with said antibody according to the invention. These couplingtechniques are well known to the person skilled in the art and will notbe expanded upon in the present description.

The invention relates, in another aspect, to a composition characterizedin that one, at least, of said antibodies, or one of their functionalfragments or derivatives, is conjugated with a cell toxin and/or aradioelement.

Preferably, said toxin or said radioelement is capable of inhibiting atleast one cell activity of cells expressing the c-Met, in a morepreferred manner capable of preventing the growth or the proliferationof said cell, especially of totally inactivating said cell.

Preferably also, said toxin is an enterobacterial toxin, especiallyPseudomonas exotoxin A.

The radioelements (or radioisotopes) preferably conjugated to theantibodies employed for the therapy are radioisotopes which emit gammarays and preferably iodine¹³¹, yttrium⁹⁰, palladium¹⁰⁰, copper⁶⁷,bismuth²¹⁷ and antimony²¹¹. The radioisotopes which emit beta and alpharays can likewise be used for the therapy.

By toxin or radioelement conjugated to at least one antibody, or one ofits functional fragments, according to the invention, it is intended toindicate any means allowing said toxin or said radioelement to bind tosaid at least one antibody, especially by covalent coupling between thetwo compounds, with or without introduction of a linking molecule.

Among the agents allowing binding in a chemical (covalent),electrostatic or noncovalent manner of all or part of the components ofthe conjugate, mention may particularly be made of benzoquinone,carbodiimide and more particularly EDC(1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide hydrochloride),dimaleimide, dithiobis-nitrobenzoic acid (DTNB), N-succinimidyl S-acetylthio-acetate (SATA), the bridging agents having one or more phenylazidegroups reacting with the ultraviolets (U.V.) and preferablyN-[-4-(azidosalicylamino)butyl]-3-(2-pyridyldithio)-propionamide (APDP),N-succinimid-yl 3-(2-pyridyldithio)propionate (SPDP),6-hydrazine-nicotinamide (HYNIC).

Another form of coupling, especially for the radioelements, can consistin the use of a bifunctional ion chelator.

Among these chelates, it is possible to mention the chelates derivedfrom EDTA (ethylenediaminetetraacetic acid) or from DTPA(diethylenetriaminepentaacetic acid) which have been developed forbinding metals, especially radioactive metals, and immunoglobulins.Thus, DTPA and its derivatives can be substituted by different groups onthe carbon chain in order to increase the stability and the rigidity ofthe ligand-metal complex (Krejcarek et al. (1977); Brechbiel et al.(1991); Gansow (1991); U.S. Pat. No. 4,831,175).

For example diethylenetriaminepentaacetic acid (DTPA) and itsderivatives, which have been widely used in medicine and in biology fora long time either in their free form, or in the form of a complex witha metallic ion, have the remarkable characteristic of forming stablechelates with metallic ions and of being coupled with proteins oftherapeutic or diagnostic interest such as antibodies for thedevelopment of radioimmunoconjugates in cancer therapy (Meases et al.,1984; Gansow et al., 1990).

Likewise preferably, said at least one antibody forming said conjugateaccording to the invention is chosen from its functional fragments,especially the fragments amputated of their Fc component such as thescFv fragments.

As already mentioned, in a preferred embodiment of the invention, saidcytotoxic/cytostatic agent or said toxin and/or a radioelement iscoupled chemically to at least one of the elements of said compositionfor simultaneous use.

The present invention comprises the described composition as amedicament.

The present invention moreover comprises the use of the compositionaccording to the invention for the preparation of a medicament.

In another aspect, the invention deals with the use of an antibody, orone of its functional fragments or derivatives, and/or of a compositionas above described for the preparation of a medicament intended toinhibit the growth and/or the proliferation of tumor cells.

Another aspect of the invention consists in the use of an antibody, orone of its functional fragments or derivatives anchor of a composition,as described above or the use above mentioned, for the preparation of amedicament intended for the prevention or for the treatment of cancer.

Is also comprised in the present invention a method intended to inhibitthe growth and/or the proliferation of tumor cells in a patientcomprising the administration to a patient in need thereof of anantibody, or one of its functional fragments or derivatives according tothe invention, an antibody produced by an hybridoma according to theinvention or a composition according to the invention.

The present invention further comprises a method for the prevention orthe treatment of cancer in a patient in need thereof, comprising theadministration to the patient of an antibody, or one of its functionalfragments or derivatives according to the invention, an antibodyproduced by an hybridoma according to the invention or a compositionaccording to the invention.

In a particular preferred aspect, said cancer is a cancer chosen fromprostate cancer, osteosarcomas, lung cancer, breast cancer, endometrialcancer, glioblastoma or colon cancer.

As explained before, an advantage of the invention is to allow thetreatment of HGF dependent and independent Met-activation relatedcancers.

In a particular aspect, the invention comprises the use of an antibody,or one of its functional fragments or derivatives and/or of acomposition, as described above or the use above mentioned, for thepreparation of a medicament intended for the prevention or for thetreatment of a patient in need thereof having a cancer characterized byoverexpression of c-Met (for example, due to a genic amplification ofc-Met) resulting in a ligand-independent constitutive activation of saidc-Met receptor.

Preferably, said cancer characterized by a genic amplification of c-Metresulting in a ligand-independent constitutive activation of said c-Metreceptor is selected from the group consisting of renal cell carcinomaand gastric cancer.

The invention, in yet another aspect, encompasses a method of in vitrodiagnosis of illnesses induced by an overexpression or anunderexpression of the c-Met receptor starting from a biological samplein which the abnormal presence of c-Met receptor is suspected, saidmethod being characterized in that it comprises a step wherein saidbiological sample is contacted with an antibody of the invention, itbeing possible for said antibody to be, if necessary, labeled.

Preferably, said illnesses connected with an abnormal presence of c-Metreceptor in said diagnosis method will be cancers.

Said antibody, or one of its functional fragments, can be present in theform of an immunoconjugate or of a labelled antibody so as to obtain adetectable and/or quantifiable signal.

The antibodies labelled according to the invention, or their functionalfragments include, for example, antibodies called immunoconjugates whichcan be conjugated, for example, with enzymes such as peroxidase,alkaline phosphatase, beta-D-galactosidase, glucose oxydase, glucoseamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malatedehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule suchas biotin, digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can belikewise conjugated to the antibodies or to their functional fragmentsaccording to the invention and especially include fluorescein and itsderivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone etc. In suchconjugates, the antibodies of the invention or their functionalfragments can be prepared by methods known to the person skilled in theart. They can be coupled to the enzymes or to the fluorescent labelsdirectly or by the intermediary of a spacer group or of a linking groupsuch as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraaceticacid (EDTA), diethylene-triaminepentaacetic acid (DPTA), or in thepresence of coupling agents such as those mentioned above for thetherapeutic conjugates. The conjugates containing labels of fluoresceintype can be prepared by reaction with an isothiocyanate.

Other conjugates can likewise include chemoluminescent labels such asluminol and the dioxetanes, bio-luminescent labels such, as luciferaseand luciferin, or else radioactive labels such as iodine¹²³, iodine¹²⁵,iodine¹²⁶, iodine¹³³, bromine⁷⁷, technetium^(99m), indium¹¹¹,indium^(113m), gallium⁶⁷, gallium⁶⁸, ruthenium⁹⁵, ruthenium⁹⁷,ruthenium¹⁰³, ruthenium¹⁰⁵, mercury¹⁰⁷, mercury²⁰³, rhenium^(99m),rhenium¹⁰¹, rhenium¹⁰⁵, scandium⁴⁷, tellurium^(121m), tellurium^(122m),tellurium^(125m), thulium¹⁶⁵, thulium¹⁶⁷, thulium¹⁶⁸, fluorine¹⁸,yttrium¹⁹⁹, iodine¹³¹. The methods known to the person skilled in theart existing for coupling the therapeutic radioisotopes to theantibodies either directly or via a chelating agent such as EDTA, DTPAmentioned above can be used for the radioelements which can be used indiagnosis. It is likewise possible to mention labelling with Na[I¹²⁵] bythe chloramine T method [Hunter W. M. and Greenwood F. C. (1962) Nature194:495] or else with technetium^(99m) by the technique of Crockford etal. (U.S. Pat. No. 4,424,200) or attached via DTPA as described byHnatowich (U.S. Pat. No. 4,479,930).

Thus, the antibody, or a functional fragment or derivative thereof,according to the invention can be employed in a process for thedetection and/or the quantification of an overexpression or of anunderexpression, preferably an overexpression, of the c-Met receptor ina biological sample, characterized in that it comprises the followingsteps:

-   -   a) the contacting of the biological sample with an antibody, or        a functional fragment or derivative thereof, according to the        invention; and    -   b) the demonstration of the c-Met/antibody complex possibly        formed.

In a particular embodiment, the antibody, or a functional fragment orderivative thereof, according to the invention, can be employed in aprocess for the detection and/or the quantification of the c-Metreceptor in a biological sample, for the monitoring of the efficacy of aprophylactic and/or therapeutic treatment of c-Met-dependent cancer.

More generally, the antibody or a functional fragment or derivativethereof, according to the invention can be advantageously employed inany situation where the expression of the c-Met receptor must beobserved in a qualitative and/or quantitative manner.

Preferably, the biological sample is formed by a biological fluid, suchas serum, whole blood, cells, a tissue sample or biopsies of humanorigin.

Any procedure or conventional test can be employed in order to carry outsuch a detection and/or dosage. Said test can be a competition orsandwich test, or any test known to the person skilled in the artdependent on the formation of an immune complex of antibody-antigentype. Following the applications according to the invention, theantibody or a functional fragment or derivative thereof can beimmobilized of labelled. This immobilization can be carried out onnumerous supports known to the person skilled in the art. These supportscan especially include glass, polystyrene, poly-propylene, polyethylene,dextran, nylon, or natural or modified cells. These supports can beeither soluble or insoluble.

By way of example, a preferred method brings into play immunoenzymaticprocesses according to the ELISA technique, by immunofluorescence, orradioimmunoassay (RIA) technique or equivalent.

Thus, the present invention likewise comprises the kits or setsnecessary for carrying out a method of diagnosis of illnesses induced byan overexpression or an underexpression of the c-Met receptor or forcarrying out a process for the detection and/or the quantification of anoverexpression or of an underexpression of the c-Met receptor in abiological sample, preferably an overexpression of said receptor,characterized in that said kit or set comprises the following elements:

-   -   a) an antibody, or a functional fragment or derivative thereof,        according to the invention;    -   b) optionally, the reagents for the formation of the medium        favorable to the immunological reaction;    -   c) optionally, the reagents allowing the demonstration of        c-Met/antibody complexes produced by the immunological reaction.

A subject of the invention is likewise the use of an antibody or acomposition according to the invention for the preparation of amedicament intended for the specific targeting of a biologically activecompound to cells expressing or overexpressing the c-Met receptor.

It is intended here by biologically active compound to indicate anycompound capable of modulating, especially of inhibiting, cell activity,in particular their growth, their proliferation, transcription or genetranslation.

A subject of the invention is also an in vivo diagnostic reagentcomprising an antibody according to the invention, or a functionalfragment or derivative thereof, preferably labelled, especiallyradiolabelled, and its use in medical imaging, in particular for thedetection of cancer connected with the expression or the overexpressionby a cell of the c-Met receptor.

The invention likewise relates to a composition as a combination productor to an anti-c-Met/toxin conjugate or radioelement, according to theinvention, as a medicament.

Preferably, said composition as a combination product or said conjugateaccording to the invention will be mixed with an excipient and/or apharmaceutically acceptable vehicle.

In the present description, pharmaceutically acceptable vehicle isintended to indicate a compound or a combination of compounds enteringinto a pharmaceutical composition not provoking, secondary reactions andwhich allows, for example, facilitation of the administration of theactive compound(s), an increase in its lifespan and/or in its efficacyin the body, an increase in its solubility in solution or else animprovement in its conservation. These pharmaceutically acceptablevehicles are well known and will be adapted by the person skilled in theart as a function of the nature and of the mode of administration of theactive compound(s) chosen.

Preferably, these compounds will be administered by the systemic route,in particular by the intravenous route, by the intramuscular,intradermal, intraperitoneal or subcutaneous route, or by the oralroute. In a more preferred manner, the composition comprising theantibodies according to the invention will be administered severaltimes, in a sequential manner.

Their modes of administration, dosages and optimum pharmaceutical formscan be determined according to the criteria generally taken into accountin the establishment of a treatment adapted to a patient such as, forexample, the age or the body weight of the patient, the seriousness ofhis/her general condition, the tolerance to the treatment and thesecondary effects noted.

Other characteristics and advantages of the invention appear in thecontinuation of the description with the examples and the figureswherein:

FIG. 1: Effect of Irrelevant IgG1 Mabs from mouse and human origin andPBS on c-Met receptor phosphorylation on A549 cells.

FIGS. 2A and 2B: Effect of murine and humanized 224G11 Mabs produced asa human IgG1/kappa isotype on c-Met receptor phosphorylation on A549cells.

-   -   FIG. 2A: agonist effect calculated as percentage versus maximal        stimulation of c-Met phosphorylation by HGF [100 ng/ml].    -   FIG. 2B; antagonist effect calculated as percentage of        inhibition of the maximal stimulation of c-Met phosphorylation        by HGF [100 ng/ml].

FIGS. 3A and 3B: Comparison between murine 224G11 Mab and chimeric224G11 Mabs containing various engineered hinge regions, on c-Metreceptor phosphorylation on A549 cells.

-   -   FIG. 3A: agonist effect calculated as percentage versus maximal        stimulation of c-Met phosphorylation by HGF [100 ng/ml].    -   FIG. 3B: antagonist effect calculated as percentage of        inhibition of the maximal stimulation of c-Met phosphorylation        by HGF [100 ng/ml].

FIGS. 4A and 4B: Comparison between murine 224G11 Mab and chimeric andhumanized 224G11 Mabs produced as a human IgG2/kappa isotype, on c-Metreceptor phosphorylation on A549 cells.

-   -   FIG. 4A: agonist effect calculated as percentage versus maximal        stimulation of c-Met-phosphorylation by HGF [100 ng/ml].    -   FIG. 48; antagonist effect calculated as percentage of        inhibition of the maximal stimulation of c-Met phosphorylation        by HGF [100 ng/ml].

FIGS. 5A and 5B: Comparison between murine 224G11 Mab and chimeric andhumanized 224G11 Mabs produced as an engineered hinge mutantTH7IgG1/kappa, on c-Met receptor phosphorylation on A549 cells.

-   -   FIG. 5A: agonist effect calculated as percentage versus maximal        stimulation of c-Met phosphorylation by HGF [100 ng/ml].    -   FIG. 5B: antagonist effect calculated as percentage of        inhibition of the maximal stimulation of c-Met phosphorylation        by HGF [100 ng/ml].

FIGS. 6A and 6B, FIGS. 7A and 7B, FIGS. 8A and 8B, FIGS. 9A and 9B,FIGS. 10A and 10G: BRET models with FIG. A: c-Met dimerization model;and FIG. B: c-Met activation model.

FIG. 11: c-Met recognition by chimeric and humanized 224G11 forms.

FIG. 12: Effect of murine and chimeric antibodies on HGF-inducedproliferation of NCI-H441 cells in vitro, NCI-H441 cells were plated inserum-free medium. 24 hours after plating m224G11 and [224G11]chim wereadded either in. absence or in presence of HGF. Black arrows indicatethe wells plated with cells alone either in absence

or in presence

of HGF. A murine IgG1 (mIgG1) was introduced as an isotype control.

FIG. 13: in vivo comparison of murine and IgG1 chimeric 224G11 Mabs onthe NCI-441 xenograft model

FIGS. 14A and 14B: Effect of the murine 224G11 Mab and of variouschimeric and humanized versions of this antibody on HGF-inducedproliferation of NCI-H441 cells in vitro. NCI-H441 cells were plated inserum-free medium. Twenty four hours after plating antibody to be testedwere added either in absence or in presence of HGF. In panel (FIG. 14A),the murine m224G11, chimeric IgG1 [224G11]chim, humanized IgG1 [224G11][Hz1], [224G11] [Hz2], [224G11] [Hz3] versions were shown. In panel(FIG. 14B), the murine m224G11 and various chimeric IgG1 forms ([224G11]chim, [224G11] [MH chim], [224G11] [MUP9H chim], [224G11] [MMCH chim],[224G11] [TH4 chim]) were presented. Black arrows indicate the wellsplated with cells alone either in absence

or in presence

of HGF. A murine IgG1 was introduced as a negative control for agonistactivity. The m5D5 was used as a dose-dependent full agonist control.

FIG. 15: Effect of the murine 224G11 Mab and of various chimeric andhumanized versions of this antibody on HGF-induced proliferation ofNCI-H441 cells in vitro. NCI-H441 cells were plated in serum-freemedium. Twenty four hours after plating antibody to be tested were addedeither in absence or in presence of HGF. The murine m224G11, [224G11]chim, [224G11] [TH7 chim]) IgG1 chimeric forms and [224G11] [TH7 Hz1],[224G11] [TH7 Hz3],) were presented. Black arrows indicate the wellsplated with cells alone either in absence

or in presence

of HGF. A murine IgG1 was introduced as a negative control for agonistactivity. The m5d5 was used as a dose-dependent full agonist control.

FIG. 16: In vivo comparison of murine, chimeric and humanized 224G11Mabs on the NCI-H441 xenograft model.

FIG. 17A: agonist effect calculated as percentage versus maximalstimulation of c-Met phosphorylation by HGF [100 ng/ml].

FIG. 17B: antagonist effect calculated as percentage of inhibition ofthe maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].

FIG. 18: BRET models with c-Met activation model.

FIG. 19: Effect on m224G11 and h224G11 on c-Met degradation on A549cells. A) Mean of 4 independent experiments +/− s.e.m. B) Western blotimage representative of the 4 independent experiments performed.

FIG. 20: Effect of m224G11 and h224G11 on c-Met degradation on NCI-H441cells. A) Mean of 4 independent experiments +/−s.c.m. B) Western blotimage representative of the 4 independent experiments performed.

FIG. 21: Set up of an ELISA to evaluate c-Met shedding.

FIG. 22: In vitro evaluation of c-Met shedding on NCI-H441 cells treatedfor days with m224G11, mlgG1 is an irrelevant antibody used as anisotype control.

FIG. 23: In vitro evaluation of c-Met shedding on amplified Hs746T,MKN45 and EBC-1 cell lines treated for 5 days with m22411. mIgG1 is anirrelevant antibody used as an isotype control. PMA is a sheddinginducer used as a positive control.

FIG. 24: In vitro evaluation of c-Met shedding on NCI-H441 and amplifiedHs746T, MKN45 and EBC-1 cell lines treated for 5 days with m224G11.mIgG1 is an irrelevant antibody used as an isotype control. PMA is ashedding inducer used as a positive control.

FIG. 25: Study of intrinsic phosphorylation of h224G11 on Hs746T cellline.

FIG. 26: Study of intrinsic phosphorylation of h224G11 on NCI-H441 cellline. A) phospho-ELISA and B) Western analysis.

FIG. 27: Study of intrinsic phosphorylation of h224G11 on Hs578T cellline. A) phospho-ELISA and B) Western analysis.

FIG. 28: Study of intrinsic phosphorylation of h224G11 on NCI-H125 cellline. A) phospho-ELISA and B) Western analysis.

FIG. 29: Study of intrinsic phosphorylation of h224G11 on T98G cellline. A) phospho-ELISA and B) Western analysis.

FIG. 30: Study of intrinsic phosphorylation of h224G11 on MDA-MB-231cell line. A) phospho-ELISA and B) Western analysis.

FIG. 31: Study of intrinsic phosphorylation of h224G11 on PC3 cell.line. A) phospho-ELISA and B) Western analysis.

FIG. 32: Study of intrinsic phosphorylation of h224G11 on HUVEC cells.

FIG. 33: In vivo comparison of the wild type murine 224G11 antibody witha chimeric hinge-engineered [224G11] [C2D5-7] Mabs on the NCI-H441xenograft model.

FIG. 34: ADCC Induction by h224G11 on both Hs746T and NCI-H441 cells.⁵¹Cr-labeled Hs746T (A) or NCI-H441 (B) cells loaded (bold squares) ornot (empty squares) with h224G11 were mixed with different ratio ofhuman NK cells and incubated for 4 hr. Cells were harvested and cpm of⁵¹Cr released by lysis was counted. The results are plotted aspercentage of lysis against the effector/target cell ratio. NL for nonloaded cells.

FIG. 35: h224G11 staining in tumor xenograft which expressed variouslevel of c-Met (A: Hs746T amplified cell line for c-Met, B: NCI-H441high level of c-Met expression and C: MCF-7 low level of c-Met).

FIG. 36: In vivo activity of h224G11 on the Hs746T xenograft model. A)FACS analysis showing c-Met expression on Hs746T cells compared to othercell lines expressing c-Met. B) QRTPCR showing c-Met amplification. C)c-Met phosphorylation in absence or in presence of HGF. D) in vivomodel.

FIG. 37: In vivo activity of h224G11 on the MKN-45 xenograft model. A)FACS analysis showing c-Met expression on MKN45 cells compared to othercell lines expression c-met. B) QRTPCR showing c-Met amplification. C)c-Met phorphorylation in absence or in presence of HGF. D) In vivomodel.

FIG. 38: In vivo activity of h224G11 on the EBC-1 xenograft model. A)FACS analysis showing c-met expression on EBC-1 cells compared to othercell lines expressing c-Met. B) QRTPCR showing c-Met amplification. C)c-met phosphorylation in absence or in presence of HGF. D) In vivomodel.

FIG. 39: IHC examination of Hs746T tumors in mice treated with theh224G11 Mab compared to control-treated tumors. (A) and (B) panelscorrespond to isotype control antibodies. (C) and (D) show theexpression of c-Met on tumor sections from mice treated with PBS orh224G11 respectively demonstrating that a chronic treatment with h224G11induced a dramatic down regulation of c-Met. Ki-67 staining issignificantly decreased on treated tumors (F) compared to PBS controlsamples (E). A significant apoptosis was observed in tumor treated withh224G11 (H) while no apoptosis was noticed in control tumors (G).

EXAMPLE 1 Generation of Antibodies Against c-Met

To generate anti-c-Met antibodies 8 weeks old BALB/c mice were immunizedeither 3 to 5 times subcutaneously with a CHO transfected cell line thatexpress c-Met on its plasma membrane (20×10⁶ cells/dose/mouse) or 2 to 3times with a c-Met extracellular domain fusion protein (10-15μg/dose/mouse) (R&D Systems, Catalog #358MT) or fragments of thisrecombinant protein mixed with complete Freund adjuvant for the firstimmunization and incomplete Freund adjuvant for the following ones.Mixed protocols in which mice received both CHO-cMet cells andrecombinant proteins were also performed. Three days before cell fusion,mice were boosted i.p. or i.v. with the recombinant protein orfragments. Then spleens of mice were collected and fused to SP2/0-Ag14myeloma cells (ATCC) and subjected to HAT selection. Four fusions wereperformed. In general, for the preparation of monoclonal antibodies ortheir functional fragments, especially of murine origin, it is possibleto refer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 726, 1988) or tothe technique of preparation of hybridomas described by Kohler andMilstein (Nature, 256:495-497, 1975).

Obtained hybridomas were initially screened by ELISA on the c-Metrecombinant protein and then by FACS analysis on A549 NSCLC, BxPC3pancreatic, and U87-MG glioblastoma cell lines to be sure that theproduced antibodies will be able to also recognize the native receptoron tumor cells. Positive reactors on these 2 tests were amplified,cloned and a set of hybridomas was recovered, purified and screened forits ability to inhibit in vitro cell proliferation in the BxPC3 model.

For that purpose 50 000 BxPC3 cells were plated in 96 well plates inRPMI medium, 2 mM L. Glutamine, without SVF. 24 after plating,antibodies to be tested were added at a final concentration ranging from0.0097 to 40 μg/ml 60 min before addition of 100 ng/ml of hHGF. After 3days, cells were pulsed with 0.5 μCi of [³H]thymidine for 16 hours. Themagnitude of [³H]thymidine incorporated into trichloroaceticacid-insoluble DNA was quantified by liquid scintillation counting.Results were expressed as raw data to really evaluate the intrinsicagonistic effect of each Mab.

Then antibodies inhibiting at least 50% cell proliferation wereevaluated for their activity on c-Met dimerization and activation BRETanalysis on transfected cells. c-Met receptor activity was quantified bymeasuring the Gab1 signalling molecule recruitment on activated c-Met.For that purpose, CHO stable cell lines expressing G-Met-Rluc orc-Met-Rluc and G-Met-K1100A-YFP for c-Met dimerization or C-Met-Rluc anda mutated form of Gab1 [Maroun et al., Mol. Cell. Biol., 1999,19:1784-1799] fused to YFP for c-Met activation were generated. Cellswere distributed in white 96 well microplates in DMEM-F12/FBS 5% culturemedium one or two days before BRET experiments. Cells were firstcultured at 37° C. with CO₂ 5% in order to allow cell attachment to theplate. Cells were then starved with 200 μl DMEM/well overnight.Immediately prior to the experiment, DMEM was removed and cells quicklywashed with PBS. Cells were incubated in PBS in the presence or absenceof antibodies to be tested or reference compounds, 10 min at 27° C.prior to the addition of coelenterazine with or without HGF in a finalvolume of 50 μl. After incubation for further 10 minutes at 37° C.,light-emission acquisition at 485 nm and 530 nm was initiated using theMithras luminometer (Berthold) (1 s/wave length/well repeated 15 times).

BRET ratio has been defined previously [Angers et al., Proc. Natl. Acad.Sci. USA, 2000, 97:3684-3689] as: [(emission at 530 nm)−(emission at 485nm)×Cf]/(emission at 485 nm), where Cf corresponds to (emission at 530nm)/(emission at 485 nm) for cells expressing Rluc fusion protein alonein the same experimental conditions. Simplifying this equation showsthat BRET ratio corresponds to the ratio 530/485 nm obtained when thetwo partners were present, corrected by the ratio 530/485 nm obtainedunder the same experimental conditions, when only the partner fused toR. reniformis luciferase was present in the assay. For the sake ofreadability, results are expressed in milliBRET units (mBU); mBUcorresponds to the BRET ratio multiplied by 1000.

After this second in vitro test, the antibody 224G11 i) withoutintrinsic activity as a whole molecule in the functional test ofproliferation, ii) inhibiting significantly BxPC3 proliferation and iii)inhibiting c-Met dimerization was selected. In the experiments, the 5D5Mab, generated by Genentech, and available at the ATCC, was added as acontrol for the intrinsic agonistic activity.

EXAMPLE 2 Humanization Process of Mouse 224G11 Mab by CDR-Grafting

1°) Humanization of the light chain variable domain (VL)

As a preliminary-step, the nucleotide sequence of 224G11 VL was comparedto the murine germline gene sequences included in the IMGT database(http://imgt.cines.fr). Murine IGKV3-5*01 and IGKJ4*01 germline genesshowing a sequence identity of 99.31% for the V region and 94.28% forthe J region, respectively, have been identified. Regarding these highhomologies, the 224G11VL nucleotide sequence has been used directly tosearch for human homologies, instead of corresponding mouse germlines.

In a second step, the human germline gene displaying the best identitywith the 224G11VL has been searched to identify the best human candidatefor the CDR grafting. For optimization of the selection, alignmentsbetween the amino acid sequences have been performed. The humanIGKV4-1*01 germline gene yielded a sequence identity of 67.30%, butshowed a different length for CDR1 (10 amino adds in 224G11 VL and 12amino acids in IGKV4-1*01). For the J region, the human IGKJ4*02germline gene (sequence identity of 77.14%) was selected.

In a next step, mouse 224G11 VL CDR regions were engrafted into theabove selected human framework sequences. Each amino acid position wasanalyzed for several criteria such as participation in VH/VL interface,in antigen binding or in CDR structure, localization of the residue inthe 3D structure of the variable domain, CDR anchors, residues belongingto the Vernier zone. Three humanized versions, corresponding to SEQ IDNo. 8, SEQ ID No. 9 and SEQ ID No. 10 were constructed, and containingrespectively four (4, 39, 40, 84), two (39, 40) or one (40) murineresidues in their PR regions and the CDRs corresponding to mouse 224G11VL.

2°) Humanization of the Heavy Chain Variable Domain (VH)

As a preliminary step, the nucleotidic sequence of the 224G11 VH wascompared to the murine germline genes sequences included in the IMGTdatabase (http://imgt.cines.fr).

Murine IGHV1-18*01, IGHD2-4*01 and IGHJ2*01 germline genes with asequence identity of 92.70% for the V region, 75.00% for the D regionand 89.36% for the J region, respectively, have been identified.Regarding these high homologies, it has been decided to use directly the224G11VH nucleotide sequences to search for human homologies, instead ofcorresponding mouse germlines.

In a second step, the human germline gene displaying the best identitywith the 224G11 VH has been searched to identify the best humancandidate for the CDR grafting. To this end, the nucleotidic sequence of224G11 VH has been aligned with the human germline genes sequencesbelonging to the IMGT database. The human IGHV1-2*02 V sequenceexhibited a sequence identity of 75.00% at the nucleotide level and64.30% at the amino acid level. Looking for homologies for the J regionled to the identification of the human IGHJ4*04 germline gene with asequence identify of 78.72%.

In a next step, mouse 224G11 VH CDR regions were engrafted into theabove selected human framework sequences. Each amino acid position wasanalysed for several criteria, such as participation in VH/VL interface,in antigen binding or in CDR structure, localization of the residue inthe 3D structure of the variable domain, CDR anchors, residues belongingto the Vernier zone. One fully humanized form, corresponding to SEQ ID 4was constructed; it contains exclusively human residues in its FRregions and the CDRs corresponding to mouse 224G11 VH.

EXAMPLE 3 Engineering of Improved Hinge Mutants

It is well known by the skilled artisan that the hinge region stronglyparticipates in the flexibility of the variable domain ofimmunoglobulins (see Brekke et al., 1995; Roux et al., 1997). during thechimerization process of 224G11 Mab, the mouse constant domain IGHG1 wasreplaced by the equivalent IGHG1 portion of human origin. Since theamino acid sequence of the hinge region were highly divergent,“murinization” of the hinge region was performed in order to keep itslength and rigidity. Since the human IGHG2 hinge region corresponds tothe closest homologue of the mouse IGHG1 hinge, this sequence was aswell considered. A series of 7 different hinge sequences wereconstructed (SEQ ID Nos. 22 to 28) by incorporating portions of themouse IGHG1 and the human IGHG2 hinges into the human IGHG1 hingeportion.

Another series of hinge mutants was designed and constructed (SEQ IDNos. 58 to 72) to evaluate the influence of either an additionalcysteine and its position along the hinge domain, deletion of 1, 2, 3 or4 amino acids along the hinge domain and a combination of these twoparameters (cysteine addition and amino acid deletion).

EXAMPLE 4 Production of Humanized 224G11 Mab and Engineered Hinge MabFormats

All above described Mab forms containing either chimeric, humanizedand/or engineered hinge regions were produced upon transienttransfection and by using the HEK293/EBNA system with pCEP4 expressionvector (InVitrogen, US).

The entire nucleotide sequences corresponding to the humanized versionsof the variable domain of 224G11 Mab light (SEQ ID No. 18, SEQ ID No. 19and SEQ ID No. 20) and heavy (SEQ ID No. 14) chains were synthesized byglobal gene synthesis (Genecust Luxembourg). They were subcloned into apCEP4 vector (InVitrogon, US) carrying the entire coding sequence of theconstant domain [CH1-Hinge-CH2-CH3] of a human IgG1 or IgG2immunoglobulin. Modification of the hinge region was performed byexchanging a {Nhel1-BCl1} restriction fragment by the equivalent portioncarrying the desired modifications, each respective {Nhe1-Bcl1} fragmentbeing synthesized by global gene synthesis (Genecust, LU). All cloningsteps were performed according to conventional molecular biologytechniques as described in the Laboratory manual (Sambrook and Russel,2001) or according to the supplier's instructions. Each geneticconstruct was fully validated by nucleotide sequencing using Big Dyeterminator cycle sequencing kit (Applied Biosystems, US) and analyzedusing a 3100 Genetic Analyzer (Applied Biosystems, US).

Suspension-adapted HEK293 EBNA cells (InVitrogen, US) were routinelygrown in 250 ml flasks in 50 ml of serum-free medium Excell 293 (SAFCBiosciences) supplemented with 6 mM glutamine on an orbital shaker (110rpm rotation speed). Transient transfection was performed with 2.10⁶cells/ml using linear 25 kDa polyethyleneimine (PEI) (Polysciences)prepared in water at a final concentration of 1 mg/ml mixed and plasmidDNA (final concentration of 1.25 μg/ml for heavy to light chain plasmidratio of 1:1). At 4 hours post-transfection, the culture was dilutedwith one volume of fresh culture medium to achieve a final cell densityof 10⁶ cells/ml. Cultivation process was monitored on the basis of cellviability and Mab production. Typically, cultures were maintained for 4to 5 days. Mabs were purified using a conventional chromatographyapproach on a Protein A resin (GE Healthcare, US).

All different forms of Mabs were produced at levels suitable withfunctional evaluations. Productivity levels are typically rangingbetween 15 and 30 mg/l of purified Mabs.

EXAMPLE 5 Evaluation of c-Met Phosphorylation Status by aPhospho-c-Met-Specific ELISA Assay

This functional assay allows to monitor modulation c-Met phosphorylationstatus either by Mabs alone or in the co-presence of HGF.

A549 cells were seeded in a 12 MW plate in complete growth medium[F12K+10% FCS]. Cells were starved for 16 hours before stimulation withHGF [100 ng/ml], and each Mab to be tested was added at its finalconcentration of 30 μg/ml 15 minutes prior to ligand stimulation.Ice-cold lysis buffer was added 15 minutes after the addition of HGF tostop the phosphorylation reaction. Cells were scaped mechanically andcell lysates were collected by centrifugation at 13000 rpm for 10 min.at 4° C. and correspond to the supernatant phase. Protein content wasquantified using a BCA kit (Pierce) and stored at −20° C. until use. Thephosphorylation status of c-Met was quantified by ELISA. A goatanti-c-Met Mab (R&D, ref AF276) was used as a capture antibody(overnight coating at 4° C.) and after a saturation step with a TBS-BSA5% buffer (1 hour at room temperature (RT)), 25 μg of protein lysateswere added to each well of the coated 96 MW plate. After a 90 minutesincubation at RT, plates were washed four time and the detectionantibody was added (anti-phospho-c-Met Mab, directed against thephopshorylated Tyr residues at position 1230, 1234 and 1235). After anadditional 1 hour incubation and 4 washes, an anti-rabbit antibodycoupled to HRP (Biosource) was added for 1 hour at RT, and theluminescence detection was performed by adding Laminol. Luminescencereadings were on a Mithras LB920 multimode plate reader (Berthold).

Both basal and HGF [100 ng/ml]-induced c-Met receptor phosphorylationlevel were unaffected neither by PBS treatment, nor by the addition ofmouse or human Mabs which do not target human c-Met receptor (FIG. 1).On the other hand, mouse (m) 224G11 Mab strongly inhibited HGF [100ng/ml]-induced c-Met phosphorylation (FIG. 2B) without altering byitself receptor phosphorylation (FIG. 2A). Surprisingly, the chimericform of 224G11 Mab (224G11chim/IgG1), meaning variable domain (VH+VL)from m224G11 combined with human constant domain IgG1/kappa yieldedstrong (17% of maximal HGF effect, FIG. 2A) agonist activity associatedwith a reduced antagonist efficacy (54% inhibition of HGF maximal effectcompared to the m224G11 that yields 75% inhibition of HGF maximumeffect, FIG. 2B). Three humanized forms of 224G11 Mab, [224G11]Hz1/IgG1,[224G11]Hz2/IgG1 and [224G11]Hz3/IgG1 also constructed on a humanIgG1/kappa backbone, yielded also decreased antagonist efficacy andsignificant agonist activity (11 to 24% of maximal HGF level) ascompared to mouse 224G11 (FIGS. 2A and 2B). A series of engineeredversions of the heavy chain hinge domain were constructed and assayed inthe c-Met receptor phosphorylation assay. As shown in FIG. 3A, animportant reduction of the agonist effect associated with the IgG1/kappaisotype was observed tor both the IgG2-based construct and forengineered IgG1/kappa constructs [MH, MUP9H and TH7]. A concomitantincrease in antagonist efficacy was as well obtained. ThehIgG1/kappa-based TH7 hinge mutant, with the most human sequence, wasselected to complete the humanization process. In a next step, threehumanized versions of 224G11 Mab variable domain were generated bycombination to either a human IgG2/kappa or an IgG1/kappa-based TH7engineered hinge constant domain. For the hIgG2/kappa humanizedconstructs, the humanized version Hz3 yielded strong agonism (FIG. 4A),and for all three humanized versions, the antagonist efficacy was belowthat observed with murine 224G11 Mab and comparable to the chimerichIgG1-based Mab (56-57% inhibition of HGF effect, FIG. 4B). On the otherhand, combination of the three humanized versions Hz1, Hz2 or Hz3 to theengineered IgG1/TH7 mutant almost fully restored the properties of mouse224G11 Mab in terms of weak agonist activity (5-6% of HGF effect) andstrong antagonist efficacy (68 to 72% inhibition of HGF effect) of c-Metreceptor phosphorylation (FIGS. 5A and 5B). These variants were highlyimproved as compared to chimeric IgG1-based 224G11 Mab but also toIgG2-based humanized forms.

A second series of engineered versions of the heavy chain hinge domainwas constructed and assayed in the c-Met receptor phosphorylation assay.As shown in FIG. 17A, all those new versions (c224G11[C2], c224G11[C3],c224G11[C5], c224G11[C6], c224G11[C7], c224G11[Δ1-3], c224G11[C7Δ6],c224G11[C6Δ9], c224G11[C2Δ5-7], c224G11[C5Δ2-6], c224G11[C9Δ2-7] andc224G11[Δ5-6-7-8]) exhibited weaker agonist effector than c224G11 sincetheir agonism activities are comprised between 6 and 14% of the HGFeffect compared to 23% for c224G11. As c224G11[TH7], all those newversions exhibited a concomitant increase in antagonist efficacy [FIG.17B]. Those results showed that engineering of the heavy chain domain bypoint mutation and/of deletion could modify agonistic/antagonisticproperties of an antibody.

EXAMPLE 6 BRET Analysis

In a first set of experiments, it had been control that irrelevant mouseIgG1, human IgG1 and human IgG2 had no effect of HGF induced BRET signalin both BRET models (representative experiment out of 12 independentexperiments; FIG. 6). These Mabs are forthwith cited as controls.

The effect of a IgG1 chimeric form of mouse 224G11 Mab ([224G11]chim) onboth c-Met dimerization and c-met activation BRET model was evaluated.While mouse 224G11 Mab inhibited 59.4% of the HGF induced BRET signal onc-Met dimerization model, [224G11]chim Mab inhibited only 28.9% (FIG.7A). [224G11]chim antibody was also less effective in inhibiting HGFinduced c-Met activation since [224G11]chim and m224G11 antibodiesinhibited respectively 34.5% and 56.4% of HGF induced BRET signal (FIG.7B). Moreover, m224G11 alone had no effect on c-Met activation while[224G11]chim had a partial agonist effect on c-Met activationcorresponding to 32.9% of the HGF induced signal. This partial agonisteffect of the [224G11]chim was also seen on c-Met dimerization BRETmodel since [224G11]chim alone induced a BRET increase corresponding to46.6% of HGF-induced signal versus 21.3% for m224G11 (FIG. 7A).

In FIGS. 8A and 8B, hinge mutated chimeric forms of 224G11 antibodyshowed a greater inhibitory effect on HGF induced BRET signal than[224G11]chim since they showed a 59.7%, 64.4%, 53.2% and 73.8%inhibition of the HGF induced activation BRET signal (FIG. 8B) and61.8%, 64.4%, 52.5% and 64.4% inhibition of the HGF induced c-Metdimerization BRET signal (FIG. 8A) for [224G11][MH chim], [224G11][MUP9Hchim], [224G11][MMCH chim] and [224G11][TH7 chim] respectively. Contraryto [224G11]chim, which had a partial agonist effect on c-Met activation,hinge mutated chimerical forms of 224G11 antibody showed no significanteffect on c-Met activation alone (5.1%, 7.6%, −2.0% and −6.9%respectively) as observed for m224G11.

In FIG. 9B, like the [224G11] [TH7 chim], the 3 humanized versions of224G11 IgG1 antibody with the TH7 hinge induced no significant increasedBRET signal in activation model when tested alone and showed a stronginhibition of HGF induced BRET signal: 59.9%, 41.8% and 57.9% for theHz1, Hz2 and Hz3 forms respectively. Moreover, [224G11] [TH7 Hz1],[224G11] [TH7 Hz2] and [224G11] [TH7 Hz3] inhibited HGF induced BRETsignal on dimerization model of 52.2%, 35.8% and 49.4% respectively(FIG. 9A).

Contrary to [224G11]chim, the chimeric form of 224G11 IgG2 antibody([224G11] [IgG2 chim]) showed no partial agonist effect alone andinhibited 66.3% of the HGF effect on the c-Met activation model (FIG.10B). On c-Met dimerization model, [224G11] [IgG2 chim] inhibited 62.4%of the HGF induced BRET signal (FIG. 10A).

The agonist efficacy of the second series of engineered versions of theheavy chain hinge domain was evaluated in c-Met activation BRET model(FIG. 18). In contrast to c224G11, which had a partial agonist effect onc-Met activation, c224G11[C2], c224G11[C3], c224G11[C5], c224G11[C6],c224G11[C7], c224G11[Δ1-3], c224G11[C7Δ6], c224G11[C6Δ9],c224G11[C2Δ5-7], c224G11[C5Δ2-6], c224G11[C9Δ2-7] and c224G11[Δ5-6-7-8]hinge mutated chimeric forms of 224G11 antibody showed no significanteffect on c-Met activation alone.

EXAMPLE 7 c-Met Recognition by Chimeric and Humanized 224G11 Forms

A direct ELISA has been set up to determine the binding ability of thevarious chimeric and humanized forms on the recombinant c-Met. Brieflyrecombinant dimeric c-Met from R&D Systems was coated at 1.25 μg/ml on96-well Immunlon II plates. After an overnight incubation at 4° C.,wells were saturated with a 0.5% gelatine/PBS solution. Plates were thenincubated for 1 hour at 37° C. before addition of 2 fold dilutions ofantibodies to be tested. Plates were incubated an additional hour beforeaddition of a goat anti-mouse IgG HRP for detecting the murine antibodyand a goat anti-human Kappa light chain HRP for chimeric and humanizedantibody recognition. Plates were incubated for one hour and theperoxydase substrate TMB Uptima was added for 5 mn before neutralizationwith H₂SO₄ IM. Results presented in FIG. 11 showed that all tested formswere comparable for c-Met recognition.

EXAMPLE 8 Effect of Murine and Chimeric 224G11 on HGF-InducedProliferation of NCI-H441 Cells in vitro

NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium(Invitrogen Corporation, Scotland, UK), 10% FCS (InvitrogenCorporation), 1% L-Glutamine (Invitrogen corporation). For proliferationassays, cells were split 3 days before use so that they were in theconfluent phase of growth before plating. NCI-H441 cells were plated in96-well tissue culture plates at a density of 3.75×10⁴ cells/well in 200μl of serum free medium (RPMI 1640 medium plus 1% L-Glutamine). Twentyfour hours after plating, antibodies to be tested were added to NCI-H441and incubated at 37° C. for thirty minutes before adding HGF at a finalconcentration of 400 ng/ml (5 nM) for 142 additional hours. The doserange tested for each antibody is from 10 to 0.0097 μg/ml (finalconcentration in each well). In this experiment, a murine IgG1 Mab wasadded as a murine isotype control and the tested antibodies were thefollowing one: m224G11 and its human IgG1 chimeric form identified as[224G11]chim. Wells plated with cells alone −/+ HGF were also included.Then cells were pulsed with 0.25 μCi of [³H]Thymidine (Amershambiosciences AB, Uppsala, Sweden) for 7 hours and 30 minutes. Themagnitude of [³H]Thymidine incorporated in trichloroaceticacid-insoluble DNA was quantified by liquid scintillation counting.Results are expressed as non transformed cpm data to better evaluate thepotential intrinsic agonist activity that could occur with anti-c-MetMabs when added alone to tumour cell.

Results described in FIG. 12 demonstrated that, as expected, the murineantibody m224G11 displayed no agonist effect when added alone to cancercells whatever the tested dose. No significant inhibition of theHBF-induced proliferation was observed with the isotype controlregarding to the cpm variations observed for this compound in thisexperiment. When added alone, the m224G11 antibody did not show anyagonist effect compared to the mlgG1 isotype control Mab or cells alone.A dose dependent anti-proliferative activities reaching 78% was observedfor m224G11 (% inhibition calculation: 100−[(cpm cells+Mab to betested−mean cpm background MIgG1)×100/(mean cpm cells +HGF−mean cpmcells alone)]). Surprisingly, the chimeric form of the 224G11 Mabsinduced a significant, dose dependent agonist effect when added alone.This agonist effect had an impact on the in vitro inhibition ofHGF-induced proliferation that shifted from 78% for the murine 224G11 to50% for its chimeric form. To determine whether such “lower” in vitrointrinsic agonist activity was compatible with an unchanged in vivoeffect, both m224G11 and [224G11]chim were produced for in vivo testing.As, in previous studies, the 30 μg/mice dose had demonstrated asignificant in vivo activity, that dose was selected for in vivoevaluation.

EXAMPLE 9

In vivo Comparison of Murine and Chimeric 224G11 Mabs on the NCI-H441Xenograft Model

NCI-H441 is derived from papillary lung adenocarcinoma, expresses highlevels of c-Met, and demonstrates constitutive phosphorylation of c-MetRTK.

To evaluate the in vivo effect of antibodies on the NCI-H441 xenograftmodel, six to eight weeks old athymic mice were housed in sterilizedfilter-topped cages, maintained in sterile conditions and manipulatedaccording to French and European guidelines. Mice were injectedsubcutaneously with 9×10⁶ cells. Then, six days after cell implantation,tumors were measurable (approximately 100 mm³), animals were dividedinto groups of 6 mice with comparable tumor size and treated first witha loading dose of 60 μg of antibody/mice and then twice a week with 30μg/dose of each antibody to be tested. The mice were followed for theobservation of xenograft growth rate. Tumor volume was calculated by theformula: π(Pi)/6×length×width×height. Results described in FIG. 13demonstrate that the murine Mab devoided of agonist activity in vivobehave, as expected, as potent antagonist even at the low tested dose.In contrast to what observed with the murine Mab, the chimeric onedisplayed a very transient in vivo activity and tumor completely escapedto the treatment at D20 post cell injection. This experimentdemonstrates clearly that the increase of in vitro agonist effect thatresulted in a decrease of antagonist activity was also responsible for asignificant in vivo loss of antagonist activity.

EXAMPLE 10 Effect of the Murine 224G11 Mab and of Various Chimeric andHumanized Versions of this Antibody on HGF-Induced Proliferation ofNCI-H441 Cells in vitro

NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium(Invitrogen Corporation, Scotland, UK), 10% FCS (InvitrogenCorporation), 1% L-Glutamine (Invitrogen Corporation). For proliferationassays, cells were split 3 days before use so that they were in theconfluent phase of growth before plating. NCI-H441 cells were plated in96-well tissue culture plates at a density of 3.75×10⁴ cells/well in 200μl of serum free medium (RPMI 1640 medium plus 1% L-Glutamine). Twentyfour hours after plating, antibodies to be tested were added to NCI-H441and incubated at 37° C. for thirty minutes before adding HGF at a finalconcentration of 400 ng/ml (5 mM) for 142 additional hours. The doserange tested for each antibody is from 10 to 0.0097 μg/ml (finalconcentration in each well). In this experiment, murine IgG1 Mab wasadded as a murine isotype control and as an agonist negative control.The tested antibodies were the following one: i) m224G11, ii) its humanIgG1 chimeric forms respectively identified as [224g11] chim, [224G11][MH chim], [224G11] [MUP9H chim], [224G11] [MMCH chim], [224G11] [TH7chim] iii) its humanized IgG1 forms respectively described as [224G11][Hz1], [224g11] [Hz2], [224G11] [Hz3]. Wells plated with cells alone −/+HGF were also included. The 5D5 whole antibody from Genentechcommercially available at the ATCC as an hybridoma cell line wasintroduced as a full agonist positive control and thereafter calledm5D5. Then cells were pulsed with 0.25 μCi of [³H]Thymidine (AmershamBiosciences AB, Uppsala, Sweden) for 7 hours and 30 minutes. Themagnitude of [³H]Thymidine incorporated in trichloroaceticacid-insoluble DNA was quantified by liquid scintillation counting.Results are expressed as non transformed cpm data to better evaluate thepotential intrinsic agonist activity that could occur with anti-c-MetMabs when added alone to tumour cell.

Results described in FIG. 14A demonstrated that as expected neither theisotype control nor the m224G11 displayed any agonist activity onNCI-H441 proliferation. The isotype control was without effect onHGF-induced cell proliferation whereas m224G11 showed a 66% inhibitionwhen added at the final concentration of 10 μg/ml. The m5D5 used as anagonist control showed, as expected, a full dose dependent agonisteffect when added alone to the cells. As already observed, the [224g11]chim Mab displayed a significant dose-dependent agonist effect and, adecreased inhibitory activity of this chimeric form was observed: 19%instead of 66% for the murine form. When added alone, the 3 IgG1humanized Mabs demonstrated dose dependent agonist effects compared tothe m224G11 form. [224G11] [Hz1], [224G11] [Hz2] and [224G11] [Hz3] hadcomparable antagonist activities about 46, 30 and 35%. These activitiesare significantly lower than the one observed for m224G11. In FIG. 14B,various IgG1 chimeric forms were tested. Compared to [224G11] chim formwhich displayed a dose-dependent agonist effect when added alone toNCI-H441 cells, the [224G1169 [MH chim], [224G11] [MUP9H chim], [224G11][MMCH chim], [224G11] TH7 chim] forms were without significant intrinsicagonist effect. Their antagonist activity was higher than the oneobserved for the m224G11 Mab (57%) with inhibitions reaching 79, 78, 84and 93% respectively for [224G11] [MH chim], [224G11] [MUP9H chim],[224G11] [MMCH chim] and [224G11] [TH7 chim].

EXAMPLE 11 In vitro Effect of Various IgG1 Humanized Form of the 224G11Mab

NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium(Invitrogen Corporation, Scotland, UK), 10% FCS (InvitrogenCorporation), 1% L-Glutamine (Invitrogen Corporation). For proliferationassays, cells were split 3 days before use so that they were in theconfluent phase of growth before plating. NCI-H441 cells were plated in96-well tissue culture plates at a density of 3.75×10⁴ cells/well in 200μl of scrum free medium (RPMI 1640 medium plus 1% L-Glutamine). Twentyfour hours after plating, antibodies to be tested were added to NCI-H441and incubated at 37° C. for thirty minutes before adding HGF at a finalconcentration of 400 ng/ml (5 nM) for 142 additional hours. The doserange tested for each antibody is from 10 to 0.0097 μg/ml (finalconcentration in each well). In this experiment, murine IgG1 Mab wasadded as a background negative control for agonist activity and thetested antibodies were the following one: i) m224G11, ii) its human IgG1chimeric forms respectively identified as [224G11] chim, [224G11] [TH4chim] iii) its humanized IgG1 forms respectively described as [224G11][TH7 Hz1], [224G11] [TH7 Hz3]. Wells plated with cells alone −/+ HGFwere also included. The 5D5 whole antibody from Genentech commerciallyavailable at the ATCC as an hybridoma cell line was introduced as a fullagonist positive control and thereafter called m5D5. Then cells werepulsed with 0.25 μμCi of [³H]Thymidine (Amersham Biosciences AB,Uppsala, Sweden) for 7 hours and 30 minutes. The magnitude of[³]Thymidine incorporated in trichloroacetic acid-insoluble DNA wasquantified by liquid scintillation counting. Results are expressed asnon transformed cpm data to better evaluate the potential intrinsicagonist activity that could occur with anti-c-Met Mabs when added aloneto tumour cell.

FIG. 15 showed that the m224G11 Mab displayed the usual inhibitoryeffect (74% inhibition). The chimeric IgG1 form [224G11] chim had asexpected a dose dependent intrinsic agonist effect and a lowerantagonist effect compared to the murine form: 33% versus 74%inhibition. The [224G11] [TH7 chim] had a very weak agonist activity inthis experiment. However it displayed a high inhibitory effect (81%)close to the one noticed for the murine Mab. The 2 humanized forms hadno intrinsic agonist effect and had an antagonist activity close to theones observed for the murine Mab or the [224G11] [TH7 chim] withrespectively 67 and 76% inhibition for [224G11] [TH7 Hz1] and [224G11][TH7 Hz3].

EXAMPLE 12 In vivo Comparison of Murine, Chimeric and Humanized 224G11Mabs Bearing Either the Wild Type or the TH7-Engineered Hinge (NCI-H441Xenograft Model)

NCI-H441 is derived from papillary lung adenocarcinoma, expresses highlevels of c-Met and demonstrates constitutive phosphorylation of c-MetRTK.

To evaluate the necessity of hinge engineering to save in vivo activityof the 224G11 murine antibody, six to eight weeks old athymic mice werehoused in sterilized filter-topped cages, maintained in sterileconditions and manipulated according to French and European guidelines.Mice were injected subcutaneously with 9×10⁶ NCI-H441 cells. Then, sixdays after cell implantation, tumors were measurable (approximately 100mm³), animals were divided into groups of 6 mice with comparable tumorsize and treated first with a loading dose of 2 mg of antibody/mice andthen twice a week with a 1 mg/dose of each antibody to be tested. Tenantibodies were evaluated in this experiment including the m224G11, thechimeric form displaying the wild type hinge (c224G11), theTH7-engineered chimeric form (224G11[TH7 chim]), three humanized formbearing the wild type hinge (224G11[IgG1 Hz1], 224G11[IgG1 Hz2] and224G11[IgG1 Hz3]) and the three corresponding TH7-engineered forms(224G11 [TH7 Hz1], 224G11[TH4 Hz2] and 224g11[TH7 Hz3]). Mice werefollowed for the observation of xenograft growth rate.

Tumor volume was calculated by the formula: π(Pi)/6×length×width×height.

Results described in FIG. 16 demonstrate that the murine Mab devoid ofany agonist activity in vitro behave, as expected, as potent in vivoantagonist. In contrast to what observed with the murine Mab, bothchimeric and humanized forms bearing the wild type hinge displayed onlya very transient in vivo activity. In any cases the substitution of thewild type hinge by the TH7-engineered one resulted in a completerestoration of the in vivo activity observed with murine antibodies.This experiment demonstrates clearly that the increase of in vitroagonist effect that resulted in a decrease of antagonist activity wasalso responsible of a significant in vivo loss of antagonist activity.It also demonstrates that the use of a TH7-engineered region instead ofthe wild type one is needed for keeping the in vivo properties of themurine Mab.

EXAMPLE 13 Effect of m224G11 and its Humanized Form h224G11 on c-MetDowregulation in vitro

In the following examples, for the avoidance of doubt, the expressionh224G11 refers to the humanized 224G11 [TH7 Hx3] of the antibody of theinvention.

Two cell lines have been selected to address the activity of anti-c-Metantibodies on c-Met receptor degradation. A549 (#HTB-174) and NCI-H441(#CCL-185) are two NSCLC cell lines from the ATCC collection. HCI-H441cells were seeded in RPMI 1640+1% L-glutamine+10% heat-inactivated FBS,at 3×10⁴ cells/cm² in six-well plates for 24 h at 37° C. in a 5% CO₂atmosphere. A549 cells were seeded in F12K+10% heat-inactivated FBS, at2×10⁴ cells/cm² in six-well plates for 24 h at 37° C. in a 5% CO₂atmosphere.

Then, cells were washed twice with phosphate buffer saline (PBS) beforebeing serum-starved for 24 additional hours. Anti-c-Met antibodies (10μg/ml), irrelevant mlgG1 (10 μg/ml) or HGF (400 ng/mL) were added inserum-free DMEM medium at 37° C. After either 4 hours or 24 hours ofincubation, the medium was gently removed and cells washed twice withcold PBS. Cells were lysed with 500 μL of ice-cold lysis buffer [50 mMTris-HCl (pB 7.5), 150 mM NaCl; 1% Nonidet P40; 0.5% deoxycholate; and 1complete protease inhibitor cocktail tablet plus 1% antiphosphatases].Cell lysates were shaken for 90 min at 4° C. and cleared at 15 000 rpmfor 10 minutes. At this stage, cell lysates could be stored at −20° C.until needed for western blot analysis. Protein concentration wasquantified using BCA. Whole cell lysates (5 μg in 20 μl) were separatedby SDS-PAGE and transferred to nitrocellulose membrane. Membranes weresaturated for 1 h at RT with TBS-Tween 20 0.1% (TBST); 5% non-fat drymilk and probed with anti-c-Met antibody (dilution 1/1000) overnight at4° C. in TBST-5% non-fat dry milk. Antibodies were diluted intris-buffered saline-0.1% tween 20 (v/v) (TBST) with 1% non-fat drymilk. Then, membranes were washed with TBST and incubated withperoxydase-conjugated secondary antibody (dilution 1:1000) for 1 h atRT. Immunoreactive proteins were visualized with ECL (Pierce # 32209).After c-Met visualization, membranes were washed once again with TBSTand incubated for 1 h at RT with mouse anti-GAPDH antibody (dilution1/200 000) in TBST-5% non-fat dry milk. Then, membranes were washed inTBST and incubated with peroxydase-conjugated secondary antibodies, for1 h at RT. Membranes were washed and GAPDH was revealed using ECL. Bandintensity was quantified by densitometry.

Results presented in FIGS. 19A and 20A demonstrated that both m224G11and h224G11 are able to significantly downregulate c-Met, in adose-dependant way, in both A549 and NCI-H441 cell lines. Thedownregulatlon is already significant after a 4 hour incubation time andstill increased at 24 hour. Histograms presented in FIGS. 19A and 20Acorresponds to mean values or respectively 4 and 3 independentexperiments. Western blot images corresponding to one significantexperiment were included in FIGS. 19B and 20B.

EXAMPLE 14 Effect of m224G11 and its Humanized Form h224G11 on c-MetShedding in vitro

Soluble shedded forms of the c-Met receptor occur naturally in the serumof mice xenografted with human tumor or in serum of human patientcarrying tumors expressing c-Met. Moreover, antibodies directed againstc-Met such as the DN30 Mab, are described as shedding inducers of c-Metin in vitro experiments. To determine whether the m224G11 as such aproperty, cells were seeded in six-well plates in 10% FCS medium. Whenthey reached approximately 80% confluence, medium was removed and freshcomplete culture medium +/− compounds to be tested was added. Cells wereincubated 72 additional hours with either m224G11, an isotype controlmIgG1 or PBS. PMA (phorbol meristate acetate) was introduced as ashedding inducer. HGF was also tested on cells to determine the impactof c-Met ligand on natural occurring shedding. Then supernatants werecollected and filtered on 0.2 μm before use in an ELISA test whichsoluble forms of c-Met were captured with an anti-c-Met antibody thatdoes not recognize the same epitope as either m224G11 or the c11E1 (FIG.21). Moreover, cells from each well were washed once with PBS and lysedto determine protein concentration. For the ELISA, 224D10 was used as acapture antibody and after plate saturation, filtered supernatants fromsix well plates were added in the ELISA test. A monomeric c-Met form wasused as a positive control. After supernatant incubation, plates werewashed to remove the unbound c-Met and c11E1 was used to detect c-Metcaptured by the 224G11 Mab. The revelation of the test was finallyperformed by addition of an HRP-conjugated anti-hFc polyclonal antibody.

Results shown in FIG. 22 indicate that a natural shedding of c-Metoccurred when cells were cultured for 72 hours in vitro. No effect ofthe mlgG1 was observed. However, the addition of m224G11 seemed toinhibit c-Met shedding. These results were confirmed for 3 other cellslines (Hs746T, EBC1 and MKN45) in FIG. 23. In that second experiment,the PMA, added as a positive shedding inducer, increased significantly,as expected, c-Met shedding at least in 2 cell lines (Hs746T and MKN45).Finally, in a third experiment (FIG. 24), HGF was introduced as acontrol. No additional shedding was induced by HGF compared as cellsalone or cells+mIgG1. Once again, a significant inhibition of c-Metshedding was observed with m224G11.

EXAMPLE 15 Intrinsic Effect of h224G11 Ab on Various Cell Lines

In previous experiments described in this patent, it has beendemonstrated that in contrast to what was observed with other antibodiessuch as 5D5, the m224G11 and its humanized form h224G11 do not displaysignificant intrinsic activity tumor cell lines. To extend this propertyto other cell lines, western blot and phospho-ELISA experiments wereperformed with the antibody alone, added for various times, on a set ofcancer cell lines, with variable levels of c-Met expression, includingHs746T, NCI-H441, Hs578T, NCI-H125, T98G, MDA-MB-231, PC3. The same testwas also performed in a normal cell: HUVEC.

Method for the phospho cMet ELISA assay was already described in example5 of the present patent application. For the western analysis, proteinlysates were made from pelleted cells by incubation in lysis buffer withproteases and phosphatase inhibitors [10 nM Tris (pH 7.4), 150 mM NaCl,1 mM sodium pyrophosphate, 1 mM EGTA, 0.5% Nonidet P40, 100 mM sodiumfluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 2 mMPMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin] at 4√ C. Protein lysateswere cleared of cellular debris by centrifugation, resolved byelectrophoresis on 8% SDS-PAGE gels, and electrotransferred to anitrocellulose membrane. For c-Met experiments, lysates wereimmunoprecipitated for specific protein of interest beforeelectrophoresis and transfer.

Results presented in FIGS. 25 to 32 demonstrate once again that nointrinsic activity of the h224G11 antibody was observed in the testedcells.

EXAMPLE 16 In vivo Comparison of the Murine Wild Type 224G11 with aChimeric Hinge-Engineered 224G11 Form Described as 224G11[C2D5-7](NCH-H441 Xenograft Model)

NCI-H441 is derived from papillary lung adenocarcinoma, expresses highlevels of c-Met and demonstrates constitutive phosphorylation of c-MetRTK.

To evaluate the necessity of hinge engineering to save in vivo activityof the 224G11 murine antibody, six to eight weeks old athymic mice werehoused in sterilized filter-topped cages, maintained in sterileconditions and manipulated according to French and European guidelines.Mice were injected subcutaneously with 9×10⁶ NCI-H441 cells. Then, sixdays after cell implantation, tumors were measurable (approximately 100mm³), animals were divided into groups of 6 mice with comparable tumorsize and treated first with a loading dose of 2 mg of antibody/mice andthen twice a week with a 1 mg/dose of each antibody to be tested. Micewere followed for the observation of xenograft growth rate. Tumor volumewas calculated by the formula: π(Pi)/6×length×width×height. Resultsdescribed in FIG. 33 demonstrate that the murine Mab devoid of anyagonist activity in vitro behave, as expected, as a potent in vivoantagonist. As suggested by the results obtained in vitro, inphosphorylation assays, the c224G11[C2D5-7] hinge-engineered antibody,that did not display a significant agonist effect, demonstrate a strongin vivo activity, comparable to the one of the m224G11 on the NCI-H441xenograft model.

EXAMPLE 17 Evaluation of H224G11 in an ADCC Test

As h224G11 is of IgG1 isotype, ADCC could be part of its in vivoefficacy in human. An in vitro [⁵¹Cr] release cytotoxicity assay wasperformed using either Hs746T or NCI-H441 cells as target cells and NKcells purified front human peripheral blood mononuclear lymphocytes.

Briefly, one million Hs746T or NCI-H441 target cells were incubated withor without 20 μg of h224G11 Ab in presence of 100 μCi of ⁵¹Chromium(Perkin Elmer) for 1 hr. Then, 4×10³ cells were plated with anincreasing number of human natural killer (NK) cells isolated fromperipheral blood mononuclear cells (PBMC) using a negative selection(Stemcell Technologies). Cells were incubated together for 4 additionalhours at 37° C. Percent of cell lysis was calculated following theformula: [(experimental ⁵¹Cr release−spontaneous ⁵¹Cr release)/(full⁵¹Cr release−spontaneous ⁵¹Cr release)]×100. Spontaneous releaserepresents the counts obtained when the target cells were cultured inabsence of natural killer cells. Full release represents the countsobtained when the target cells were lysed with 1% Triton X-100 h224G11significantly enhanced lysis of both Bs746T (FIG. 34 a) and NCI-H441(FIG. 34 b) cells by 62.9% and 63.2%, respectively, at a ratio NK/targetcells of 100.

EXAMPLE 18 Immunohistochemical Studies (IHC)

Procedures of Paraffin Embedded Tumors IHC Staining: 8 to 12 μM sectionsof frozen tumor were and immediately fixed in pre cooled acetone −20° C.for 3 minutes. Slides were then cooled at room temperature for 30minutes to 1 hour. After 2 washes in PBS the Endogenous peroxidaseactivity was blocked using Peroxidase Blocking Reagent (Dako K4007) forfive minutes. Sections were washed with PBS and incubated inavido/biotin blocking reagent (Dako X0590) just before saturation of thenon specific sites in PBS-BSA 4% for 30 minutes at room temperature.Then, slides were incubated with the biotinylated h224G11 (50 to 10μg/ml) or human biotinylated IgG1/kappa (50 to 10 μg/ml, the BindingSite) as negative control 2 hours at room temperature.

Sections were washed with PBS and incubated with Streptavidin-peroxydasecomplex universal (Dako K0679) for 30 to 45 minutes.3-Amino-9-Ethylcarbozole was used for development of a red reactionproduct (Sigma). The slides were immersed in hematoxylin for 4 minutesto counterstain (Dako S3309).

Results are represented in FIG. 35.

h224G11 differentially stains the cell membrane of various tumor types.In this immunohistochemistry procedure, the red reaction productcorrelates to positive staining of the cell membrane and lack of redreaction product correlates to negative staining and no visualization ofthe cell membrane. The IgG control, human IgG1/kappa is an isotypematched control.

EXAMPLE 19 In vivo Activity of anti-c-Met Mabs on Three Cell LinesAmplified for c-Met and Xenografted in Mice

Hs746T (ATCC) and MKN45 (JCRB), two stomach carcinoma cells, and, EBC-1(JCRB), a lung squamous cell carcinoma, are 3 cell lines thatover-express c-Met as shown by FACS analysis in FIGS. 36A, 37A and 38A.These cell lines are also described in the literature as amplified celllines for c-Met. To confirm this latter point, c-Met gene amplificationwas determined by quantitative real-time PCR using ΔΔCt method. Fifty ngof genomic DNA from each indicated cell line, extracted using the QIAampDNA midi kit (Qiagen, Les Ulis, FR) was used. The reference gene was DNAtopoisomerase III alpha located on chromosome 17. Primers for c-Met genewere located on exon 14. The corresponding primers for both genes arethose described by Smolen et al. (PNAS, 2000, 103:2316-2321) and yieldedsimilar PCR efficiencies. The QPCR protocol consists in a 10 min cycleat 95° C., followed by 40 successive cycles of 15 sec at 95° C./45 secat 95° C. Data were first normalized to the reference gene, and theneach cell line was normalized to MCF-7 cells containing a single copy ofc-Met gene per haploid genome. The results presented in FIG. 36B, 37Band 38B demonstrate that the 3 cell lines display a significantamplification of c-Met ranking from 5 to 10-fold gene amplification.

As it has been described that such cells are constitutively activatedfor c-Met, a Western blot assay was performed to determine the level ofc-Met phosphorylation of each cell line either in absence or in presenceof HGF (50 ng/ml). For that purpose cells were cultured overnight inserum-free medium and then treated or not with 50 ng/ml of HGF for 2min. Cells were lysed with lysis buffer supplemented with proteaseinhibitors. Cells lysates were incubated at 4° C. for 90 min., thencentrifuged and supernatants were collected. Protein concentrations weredetermined using the BCA protein assay (Pierce) and samples wereimmunoblotted. Antibodies used include the anti-c-Met antibody, clone25H2 from Cell Signaling and the anti-P-c-Met Y1230, 1234, 1235 fromBiosource. Immunoblotting with the phospho-specific c-Met antibodyagainst Y1230/1234/1235 showed strong baseline phosphorylation of thereceptor in each amplified cell line (FIGS. 36C, 37C and 38C). Noadditional phosphorylation is observed when HGF was added to thesecells. This last observation confirms that c-Met was activated in anHGF-independent way in Hs746T, MKN45 and EBC-1 cell lines. on

In order to test our anti-c-Met antibodies in vivo on ligand-independentcell lines, Hs746T, MKN45 and EBC-1 cells were routinely cultured incompleted DMEM, RPMI 1640 or EMEM medium respectively. Cells were splittwo days before engraftment so that they were in exponential phase ofgrowth. Seven million cells were engrafted to either SCID (for Hs746Tand EBC-1) or CD1 Swiss nude nice (for MKN45). Five to seven days afterimplantation, tumors were measured and animals were divided into groupsof 6 mice with comparable tumor size. Mice were treated i.p. with aloading dose of 2 mg of h224G11 Mab/mouse and then twice a week with 1mg of antibody/mouse. Tumor volume was measured twice a week andcalculated by the formula: π/6×length×width×height. Statistical analysiswas performed at each measured time using a Mann-Whitney test.

This experiment demonstrates that h224G11 reduced significantly tumorgrowth of Hs746T, MKN45 and EBC1 (FIGS. 36D, 37D and 38D), indicatingthat the humanized antibody is able to inhibit tumor growth of gastricand lung ligand-independent cell lines. Before these results wereobtained, it would not have been apparent how the presence of themodified hinge in this humanized antibody would affect itsligand-independent inactivation of c-Met, because the altered hinge alsoaffected its agonist/antagonist properties.

EXAMPLE 20 Ex vivo Analysis of h224G11 Activity on the Hs746T XenograftModel in SCID Mice

In order to determine more precisely mechanisms of action that resultedin Hs746T tumor xenograft inhibition, the effect of 3. i. p. injectionsof h224G11 was evaluated on tumor mitotic index (Ki67), apoptosis(activated caspase 3) and c-Met expression using immunohistochemistry(IHC) methods. Three mice were analyzed for each condition. Asignificant down regulation of c-Met was observed on treated tumorscompared to control one (FIGS. 39C and D respectively). Panel 39A and Bcorresponds to isotypes controls. In addition a significant decrease inKi67 levels (FIGS. 39E and F) and an increase in activated caspase 3staining (FIGS. 39G and H) was noticed on all treated tumors indicatingthat h224G11 was able to inhibit significantly cell proliferation and toinduce cell apoptosis within the tumor.

1. A monoclonal antibody, or a divalent functional fragment orderivative thereof capable to inhibit the c-Met dimerization, saidantibody comprising a heavy chain comprising: CDR-H1, CDR-H2 and CDR-H3with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3; and alight chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively theamino acid sequences SEQ ID Nos. 5, 6 and 7, said antibody being furthercharacterized in that it also comprises a hinge region comprising theamino acid sequence SEQ ID No.
 56. 2. The antibody of claim 1, or adivalent functional fragment or derivative thereof characterized in thatsaid hinge region comprises the amino acid sequence SEQ ID No.
 57. 3.The antibody of claim 1, or a divalent functional fragment or derivativethereof characterized in that said hinge region comprises the amino acidsequence SEQ ID No.
 21. 4. The antibody of claim 1, or a divalentfunctional fragment or derivative thereof characterized in that saidhinge region comprises an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 22 to 28 and SEQ ID Nos. 72 to
 86. 5. Theantibody of claim 1, or a divalent functional fragment or derivativethereof characterized in that it consists of a chimeric antibody.
 6. Theantibody of claim 1, or a divalent functional fragment or derivativethereof, characterized in that it consists of a human antibody.
 7. Theantibody of claim 1, or a divalent functional fragment or derivativethereof, characterized in that it consists of a humanized antibody. 8.The antibody of claim 7, or a divalent functional fragment or derivativethereof, characterized in that it comprises a heavy chain variabledomain of sequence comprising the amino acid sequence SEQ ID No. 4; anda light chain variable domain of sequence comprising the amino acidsequence SEQ ID No. 8, 9 or
 10. 9. The antibody of claim 8, or adivalent functional fragment or derivative thereof, characterized inthat it comprises a heavy chain variable domain of sequence comprisingthe amino acid sequence SEQ ID No. 4; a light chain variable domain ofsequence comprising the amino acid sequence SEQ ID No. 8; and a hingeregion comprising the amino acid sequence SEQ ID No.
 22. 10. Theantibody of claim 8, or a divalent functional fragment or derivativethereof, characterized in that it comprises a heavy chain variabledomain of sequence comprising the amino acid sequence SEQ ID No. 4; alight chain variable domain of sequence comprising the amino acidsequence SEQ ID No. 9; and a hinge region comprising the amino acidsequence SEQ ID No.
 22. 11. The antibody of claim 8, or a divalentfunctional fragment or derivative thereof characterized in that itcomprises a heavy chain variable domain of sequence comprising the aminoacid sequence SEQ ID No. 4; a light chain variable domain of sequencecomprising the amino acid sequence SEQ ID No. 10; and a hinge regioncomprising the amino acid sequence SEQ ID No.
 22. 12. The antibody ofclaim 8, or a divalent functional fragment or derivative thereofcharacterized in that it comprises a heavy chain variable domain ofsequence comprising the amino acid sequence SEQ ID No. 4; a light chainvariable domain of sequence comprising the amino acid sequence SEQ IDNo. 8; and a hinge region comprising the amino acid sequence SEQ ID No.28.
 13. The antibody of claim 8, or a divalent functional fragment orderivative thereof, characterized in that it comprises a heavy chainvariable domain of sequence comprising the amino acid sequence SEQ IDNo. 4; a fight chain variable domain of sequence comprising the aminoacid sequence SEQ ID No. 9; and a hinge region comprising the amino-acidsequence SEQ ID No.
 28. 14. The antibody of claim 8, or a divalentfunctional fragment or derivative thereof characterized in that itcomprises a heavy chain variable domain of sequence comprising the aminoacid sequence SEQ ID No. 4; a light chain variable domain of sequencecomprising the amino acid sequence SEQ ID No. 10; and a hinge regioncomprising the amino acid sequence SEQ IB No.
 28. 15. An isolatednucleic acid, characterized in that it is chosen from the followingnucleic acids: a) a nucleic acid, DNA or RNA, coding for an antibody, ora divalent functional fragment or derivative thereof as claimed in oneof claims 1 to 14; b) a nucleic acid comprising a DNA sequencecomprising the sequences SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 andthe sequences SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17; c) anucleic acid comprising a DNA sequence comprising the sequences SEQ IDNo. 14 and SEQ ID No. 18, 19 or 20; d) the corresponding RNA nucleicacids of the nucleic acids as defined in b) or c); e) the complementarynucleic acids of the nucleic acids as defined in a), b) and c); f) anucleic acid of at least 18 nucleotides capable of hybridizing underconditions of high stringency with at least one of the CDRs of sequenceSEQ ID Nos. 11 to 13 and 15 to
 17. 16. An isolated nucleic acid,characterized in that it is chosen from the following nucleic acids: anucleic acid, DNA or RNA, coding for an antibody, or one of itsfunctional fragments or derivatives, according to the present inventionand wherein the nucleic sequence coding for the hinge region of saidantibody comprises or has a sequence selected from the group consistingof the sequences SEQ ID Nos. 29 to 35 and SEQ ID Nos. 73 to
 87. 17. Avector comprising a nucleic acid as claimed in claim 15 or
 16. 18. Ahost cell comprising a vector as claimed in claim
 17. 19. A transgenicanimal with the exception of man comprising at least one celltransformed by a vector as claimed in claim
 17. 20. A process forproduction of an antibody, or a divalent functional fragment orderivative thereof, as claimed in one of claims 1 to 14, characterizedin that it comprises the following stages: a) culture in a medium andappropriate culture conditions of a cell as claimed in claim 18; and b)the recovery of said antibody, or a divalent functional fragment orderivative thereof, thus produced starting from the culture medium orsaid cultured cells.
 21. An antibody, or a divalent functional fragmentor derivative thereof, capable of being obtained by a process as claimedin claim
 20. 22. The antibody of claims 1 to 14 and 21 as a medicament.23. A composition comprising by way of active principle a compoundconsisting of an antibody, or a divalent functional fragment orderivative thereof, as claimed in one of claims 1 to 14, 21 or
 22. 24.The composition of claim 23, characterized in that it comprises,moreover, as a combination product for simultaneous, separate ofsequential use, an anti-tumoral antibody.
 25. The composition of claim23, characterized in that it comprises, moreover, as a combinationproduct for simultaneous, separate or sequential use, acytotoxic/cytostatic agent.
 26. The composition of claim 23,characterized in that one, at least, of said antibodies, of a divalentfunctional fragment or derivative thereof, is conjugated with a celltoxin and/or a radioelement.
 27. The composition of claim 25 or 26,characterized in that said cytotoxic/cytostatic agent or said toxinand/or a radioelement is coupled chemically to at least one of theelements of said composition for simultaneous use.
 28. The compositionas claimed in one of claims 23 to 27 as a medicament.
 29. The use of anantibody, or a divalent functional fragment or derivative thereof, ofclaims 1 to 14, 21 or 22 or of a composition of claims 23 to 28 for thepreparation of a medicament intended to inhibit the growth and/or theproliferation of tumor cells.
 30. The use of an antibody, or a divalentfunctional fragment or derivative thereof, of claims 1 to 14, 21 or 22or of a composition of claims 23 to 28, or the use of claim 29, for thepreparation of a medicament intended for the prevention or for thetreatment of cancer.
 31. The use of claim 30, characterized in that saidcancer is a cancer chosen from prostate cancer, osteosarcomas, lungcancer, breast cancer, endometrial cancer, glyoblastoma or colon cancer.32. The use of claim 30 or 31, characterized in that said cancer is aHGF dependant and independent Met-activation related cancer.
 33. The useas defined by claim 30, for the prevention or the treatment of a patientin need thereof having a cancer characterized by ligand-independentactivation of c-Met.
 34. The use as defined by claim 30, for theprevention or the treatment of a patient in need thereof having a cancercharacterized by overexpression of c-Met.
 35. The use as defined byclaim 30 for the prevention or the treatment of a patient in needthereof having a cancer characterized by overexpression of c-Metresulting from genic amplification of c-Met.
 36. The use as defined byclaim 35, for the prevention or the treatment of a patient in needthereof having a cancer characterized by overexpression of c-Metresulting from genic amplification of c-Met and resulting inligand-independent activation of c-Met.
 37. The use of claim 35, whereinsaid cancer is selected from the group consisting of renal cellcarcinoma and gastric cancer.
 38. The use of claim 36, wherein saidcancer is selected from the group consisting of renal cell carcinoma andgastric cancer.
 39. A method of in vitro diagnosis of illnesses inducedby an overexpression or an underexpression of the c-Met receptorstarting from a biological sample in which the abnormal presence ofc-Met receptor is suspected, characterized in that said method comprisesa step wherein said biological sample is contacted with an antibody ofclaims 1 to 14, 21 or 22, it being possible for said antibody to be, ifnecessary, labelled.